JP2023543604A - Host cells that overexpress translation factors - Google Patents

Abstract

A recombinant eukaryotic host cell expressing a gene of interest (GOI), wherein the gene is modified to enhance the expression of two or more genes encoding messenger ribonucleoprotein (mRNP) translation initiation factors (TIF genes). , the TIF gene comprises at least a gene encoding eIF4A and a gene encoding eIF4G; A recombinant eukaryotic host cell, wherein expression of at least one of the GOIs is under the transcriptional control of a promoter different from the promoter that controls expression of the GOI. [Selection diagram] None

Description

The present invention refers to improving the yield of recombinant protein production and host cells engineered to increase the expression of one or more translation factors.

Proteins produced in recombinant host cell culture are becoming increasingly important as diagnostic and therapeutic agents. To this end, cells are engineered and/or selected to produce abnormally high levels of the recombinant or heterologous protein of interest.

Successful production of proteins of interest (POI) has been achieved in eukaryotic host cells in cell culture. Eukaryotic host cells, especially mammalian host cells, yeast or fungi, or bacteria are commonly used as production hosts for biopharmaceutical proteins and bulk chemicals. The most prominent examples are methylotrophic yeasts such as Pichia pastoris, which are well known for their efficient secretion of heterologous proteins. In 2005, P. pastoris has been reclassified into a new genus, Komagataella, and K. pastoris has been reclassified into a new genus, Komagataella. pastoris, K. phaffii, K. pseudopastoris was divided into three species. Strains commonly used for biotechnological applications are K. pastoris and K. pastoris. phaffii belong to two proposed species. Strains GS115, X-33, CBS2612, and CBS7435 were derived from K. phaffii, but strain DSMZ70382 is of the type species K. phaffii. pastoris, which includes all available P. pastoris. pastoris strain (Kurtzman 2009, J Ind Microbiol Biotechnol. 36(11):1435-8). Mattanovich et al. (Microbial Cell Factories 2009, 8:29 doi:10.1186/1475-2859-8-29), K. We describe the genome sequencing of the type strain DSMZ70382 of P. pastoris and analyze its secretome and sugar transporters.

Ribosomes are complex ribonucleoprotein assemblies that perform protein synthesis. Messenger ribonucleoproteins (mRNPs) are mRNA-protein complexes in which transcripts are bound by a changing series of proteins that mediate the co-transcriptional and post-transcriptional events that make up the transcript life cycle. Transcripts initially undergo 5' end capping, often splicing, 3' cleavage and polyadenylation, mRNA quality control by nuclear exosomes, and recruitment of transport factors. They are then transported into the cytoplasm and some undergo specific intracellular localization. Transcripts are ultimately translated and degraded, often in a controlled manner.

Translation initiation is an important pathway for the production of recombinant proteins. The formation of a closed-loop structure consisting of mRNA, some eukaryotic initiation factors (eIFs), and ribosomal proteins has been considered to assist in the initiation of translation and thus improve overall translation efficiency.

Mead et al. (Biochem. J. 2015, 472: 261-273) across a panel of 30 recombinant CHO cell lines producing monoclonal antibodies (mAbs), the key components of the closed loop, eIF (eIF3a, eIF3b, eIF3c, eIF3h, eIF3i, and eIF4G1), poly(A)-binding protein (PABP) 1 and PABP-interacting protein 1 (PAIP1) mRNA and protein levels are described. High-producing cell lines were found to maintain amounts of translation initiation factors involved in closed-loop mRNA formation and maintain these proteins at appropriate levels to provide enhanced recombinant protein production. .

The eIF4F complex consists of the cap binding proteins eIF4E, eIF4G, and the RNA helicase eIF4A. eIF4G is a scaffold protein that has binding domains for PABP (PAB1), eIF4E, eIF4A, and (in mammals) eIF3. Yeast and human eIF4G also bind RNA. The binding domains of eIF4E and PABP in eIF4G, along with its RNA binding activity, allow eIF4G to coordinate independent interactions with mRNA through the cap, poly(A) tail, and mRNA body sequences to create a "closed loop" It allows for the assembly of stable circular messenger ribonucleoproteins (mRNPs), called structures.

The closed-loop model proposes the interaction of the 5' and 3' ends of the mRNA through a cross-linking mechanism mediated by several proteins, including several translation initiation factors. The closed-loop core bridge is between the 5' cap, eIF4F (consisting of eIF4A, eIF4E, and eIF4G), eIF3, poly(A)-binding protein (PABP)-interacting protein 1 (PAIP1), PABP1, and the poly(A) tail. is formed. This circularization of the mRNA may be achieved by enhancing ribosome recycling and/or ensuring that eIF4F remains tethered to the mRNA and does not need to be remobilized from the free eIF4F pool with each round of translation initiation. It is almost accepted to increase translation speed by The elongation, termination, and recycling steps of translation in eukaryotes are described by Dever et al. (Cold Spring Harb Perspect Biol 2012;4:a013706) and Hinnebusch et al. 011544).

Roobol et al. (Metabolic Engineering 2020, 59:98-105) showed that transient and stable overexpression of the eIF3i and eIF3v subunits of the large eIF3 complex in mammalian cell lines HEK and CHO cell lines resulted in increased proliferation rates, respectively. , investigating the effects on increased protein synthesis capacity and delayed apoptosis. eIF3i is a eukaryote that contains a single copy of 12 different subunits, of which 5 (a, b, c, g, and i) are conserved and essential in vivo from yeast to mammals. It is a component of the initiation factor 3 (eIF3) complex.

Archer et al. (RNA Biol. 2015 Mar; 12(3):248-254) investigated the mRNA closed loop formed by the interaction between the cap structure, poly(A) tail, eIF4E, eIF4G, and PAB in yeast.

Chan et al. (eLife 2018;7:e32536) describe that inhibition of translation initiation destabilizes individual transcripts and accelerates mRNA degradation in yeast. Overexpression of the 5' cap-binding mutant of eIF4E caused a subtle inhibition of proliferation. A strong synthetic proliferation defect was observed when eIF4E and eIF4G were downregulated simultaneously.

Translation initiation factors eIF4E, eIF4G1, and eIF4G2, which are present in the 39S and 57S translation complexes, are copurified with PAB1. Such a complex contains the closed-loop factors eIF4E, eIF4G, and PAB1 and apparently associates with mRNA through binding of eIF4E to the mRNA cap and PAB1 to the polyadenylation tail (Denis et al. Nature Scientific Reports 2018 , 8:11468).

RLI1 is known to be important for ribosome recycling, required for efficient stop codon recognition, and thus stimulates translation termination. However, RLI1 has dual functions in translation initiation and ribosome biogenesis. Yarunin et al. (EMBO Journal 2005, 24:580-588) describe that RLI1 has a function in ribosome formation associated with pre-40S particles and mature 40S subunits. RLI1 is specifically associated with MFC components and the 40S ribosome (Dong et al. THE JOURNAL OF BIOLOGICAL CHEMISTRY 2004, 279(40):42157-42168).

Liao et al. (Biotechnol Lett (2020). https://doi.org/10.1007/s10529-020-02977-z) disclosed the expression profile of eGFP under methanol induction in a translation-related factor overexpression strain. We identified Bcy1, a ribosome biogenesis factor, as a factor that significantly increases eGFP expression when overexpressed under methanol induction. eIF4A and eIF4G overexpressors had no significant effect in such expression systems. Bcy1 is a regulatory subunit of cyclic AMP-dependent protein kinase (PKA), which regulates ribosomal protein genes, postdiauxic shift genes, and stress response element genes, and regulates cell proliferation and heterologous protein expression. results in improvements.

WO2019/173204A1 discloses yeast for use in large-scale ethanol production that overexpresses PAB1, thereby reducing acetate formation. US5646009 contains a DNA segment encoding eIF4E and another DNA segment encoding a protein of interest in one open reading frame, thereby increasing the expression of the protein in eukaryotic host cells, particularly HEK cells. Discloses a hybrid vector.

CN110551750 refers to improving the efficiency of yeast mRNA expression by overexpressing RLI1.

EP3663319 discloses expressing a fusion protein comprising PAB1 and eIF4G to express the protein of interest in yeast.

This Summary is provided to introduce a selection of concepts in a simplified form that are further described below in the Detailed Description. This Summary is not intended to identify key or essential features of the claimed subject matter or to limit the scope of the claimed subject matter. Other features, details, utilities, and advantages of the claimed subject matter will be apparent from the following written detailed description, including those aspects that are illustrated in the accompanying drawings and defined in the appended claims. It will become clear.

The purpose of the present invention is to improve the production of recombinant proteins in production host cells. A particular aim is to increase the yield of recombinant proteins by increasing translation efficiency.

This object is solved by the subject matter of the claims and is further described herein.

The present invention provides recombinant eukaryotic host cells that express a gene of interest (GOI), which, compared to the host cell prior to one or more genetic modifications, has a messenger ribonucleoprotein (mRNP) TIF genes are engineered by genetically modifying them to increase the expression of two or more genes encoding translation initiation factors (TIF genes).

Specifically, two of the more TIF genes are TIF genes that include at least a gene encoding eIF4A and a gene encoding eIF4G.

According to certain embodiments, the TIF gene further comprises any one or more of the genes encoding eIF4E, PAB1, or RLI1.

According to a particular embodiment, the TIF gene encodes a TIF comprising or consisting of the following TIFs, in particular comprising or consisting of a combination of the following TIFs:
a) eIF4A and eIF4G,
b) eIF4A, eIF4G, and eIF4E,
c) eIF4A, eIF4G, eIF4E, and PAB1,
d) eIF4A, eIF4G, and PAB1.

In particular, any of the TIF combinations of embodiments a) to d) above may optionally further include RLI1.

According to certain embodiments, the host cell is engineered to overexpress at least:
a) Genes encoding eIF4A and eIF4G,
b) genes encoding eIF4A, eIF4G, and eIF4E;
c) genes encoding eIF4A, eIF4G, eIF4E, and PAB1;
d) Genes encoding eIF4A, eIF4G, and PAB1.

Specifically, the host cell is engineered to overexpress any of the gene combinations listed in a) to d) above, and optionally engineered to overexpress RLI1. can be further manipulated to Specifically, the expression of at least one of the TIF genes is under the transcriptional control of a promoter different from the promoter that controls the expression of the GOI. Specifically, GOI is expressed by a GOI expression cassette (GOIEC) and each TIF gene is expressed by a TIF gene expression cassette (TIFEC). The expression cassette contains or consists of at least a promoter operably linked to the gene to be expressed.

According to certain embodiments, the GOIEC promoter is different from any one or more or all of the TIFEC promoters.

Promoters are specifically included in each separate expression cassette to express the TIF gene and GOI.

Specifically, the TIF of mRNP is, for example, the TIF that is present in the mRNP complex or activated mRNP prior to binding to the 43S preinitiation complex (PIC). Among such TIFs are, in particular, one or more closed-loop factors such as eIF4E, eIF4A, eIF4G, PAB1, and/or RLI1, which are understood to be associated with closed-loop structures, in particular eIF4E, eIF4A, or There is one or more of the factors of the eIF4F complex, such as eIF4G.

Specifically, the eIF4G is preferably eIF4G2 (TIF4632, eukaryotic initiation factor 4F subunit p130) of yeast such as Pichia or Saccharomyces.

According to certain embodiments, the host cell is genetically engineered to overexpress at least two, at least three, at least four, or at least five of the TIF genes, and the TIF genes are at least Contains genes encoding eIF4A and eIF4G.

Specifically, at least two of the TIFs are of the eIF4F complex (particularly eIF4A, eIF4G, and optionally eIF4E). Specifically, two, three, or more or all of the TIF genes of the eIF4F complex (particularly eIF4A, eIF4G, and optionally eIF4E) are overexpressed.

In particular, at least one of the TIFs may be a closed loop factor, in particular eIF4G, and optionally either one or both of eIF4E or PAB1. In particular, one, two, three or more or all closed loop factors (in particular eIF4G and optionally either one or both of eIF4E or PAB1) are overexpressed. .

Specifically, the TIF gene may be of eukaryotic or prokaryotic (particularly yeast or mammalian) origin and may be a naturally occurring gene or an artificial variant thereof, in particular a naturally occurring TIF (naturally occurring isotope). or a functionally active variant that has high sequence identity and has approximately the same or increased function as TIF in activated mRNP and/or translation initiation in the production host cell. include.

Specifically, the TIF gene is of eukaryotic origin, eg, from any of the host cell types further described herein. For example, from:
a) Yeast cells of a genus selected from the group consisting of Pichia, Hansenula, Komagataella, Saccharomyces, Krivellomyces, Candida, Ogataea, Yarrowia, and Geotrichum (preferably Pichia pastoris, Komagataella phaffii, Komagataella pastoris, Komagataella pseudopastoris, Saccharomyces cerevisiae, Ogataea minuta, Kluyveromces lactis, Kluyveromes marxianus, Yarrowia lipolytica, or Hansenula polymorpha), or b) cells of filamentous fungi (e.g., Aspergillus awamori or Trichoderma reesei), or c) non-human primates. , human, rodent, or bovine cells (e.g., mouse myeloma (NSO) cell line, Chinese hamster ovary (CHO) cell line, HT1080, H9, HepG2, MCF7, MDBK, Jurkat, MDCK, NIH3T3, PC12, BHK (baby hamster kidney cells), VERO, SP2/0, YB2/0, Y0, C127, L cells, COS (COS1 and COS7, etc.), QC1-3, HEK-293, VERO, PER.C6, HeLA, EBI, EB2, EB3, oncolytic or hybridoma cell lines), or d) insect cells (e.g. Sf9, Mimic™ Sf9, Sf21, High Five (BT1-TN-5B1-4), or BT1-Ea88 cells), or e) an algal cell (e.g., Amphora, Tridia, Dunaliella, Chlorella, Chlamydomonas, Cyanobacteria, Nannochloropsis, Spirulina, or Ochromonas), or f) a plant cell ( For example, cells derived from monocotyledonous plants (preferably corn, rice, wheat, or hackberry) or cells derived from dicot plants (preferably cassava, potato, soybean, tomato, tobacco, alfalfa, Physcomitrella patens, or Arabidopsis). cell).

According to certain embodiments, the TIF gene is of eukaryotic origin, eg, derived from the same species origin as the POI. In particular, the TIF gene may be of yeast origin (eg Pichia or Saccharomyces) or of mammalian origin (eg human or non-human animal origin). According to certain embodiments, any one, two, three, four, or five of the TIFs are yeast or mammalian (e.g., human, mouse, hamster, or ape) proteins (naturally occurring isotopes). (including forms).

According to certain embodiments, either the yeast or human TIF gene is selected for overexpression in the host cell.

Specifically, TIFs used for the purposes provided herein are 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% %, or 100% of the sequence of each naturally occurring (also referred to as native or wild type) TIF (particularly of the TIF identified by the sequences provided herein). comprising or consisting of an amino acid sequence identical to any one of the following:

According to certain aspects,
a) The TIF gene encodes eIF4E and has any one of SEQ ID NOs: 1 to 11 and 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98 %, 99%, or 100% sequence identity;
b) The TIF gene encodes eIF4A and has any one of SEQ ID NOs: 12 to 33 and 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98 %, 99%, or 100% sequence identity;
c) The TIF gene encodes eIF4G and has any one of SEQ ID NOs: 34 to 44 and 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98 %, 99%, or 100% sequence identity;
d) The TIF gene encodes PAB1 and has any one of SEQ ID NOs: 45 to 55 and 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98 %, 99%, or 100% sequence identity, and e) the TIF gene encodes RLI1 and comprises any one of SEQ ID NOs: 56-65. at least one of 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with one of Contains or consists of.

According to certain aspects,
a) The eIF4E protein is 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or comprising or consisting of at least one of 100% sequence identity;
b) the eIF4A protein is 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or comprising or consisting of at least one of 100% sequence identity;
c) eIF4G protein is 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or comprising or consisting of at least one of 100% sequence identity;
d) The PAB1 protein has any one of SEQ ID NOs: 45 to 55 and 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity;
e) RLI1 protein is 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or comprises or consists of at least one of 100% sequence identity.

Specifically, a TIF gene used for the purposes provided herein is a nucleic acid molecule comprising or consisting of a nucleotide sequence encoding the respective TIF. A TIF gene may comprise or consist of a naturally occurring (also referred to as native or wild-type) nucleotide sequence or may be mutated (e.g., optimized for expression of the TIF gene in a host cell). (eg, a codon-optimized sequence or a Golden Gate-optimized sequence).

In particular, the TIF genes used for the purposes provided herein are those that differ from the sequence of the respective naturally occurring (also referred to as native or wild-type) TIF gene (particularly as defined herein). with any one of the TIF genes identified by the sequences provided in ), 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical.

According to certain embodiments, the one or more genetic modifications include (i) one or more polynucleotides or portions thereof, or (ii) expression control sequences, preferably promoters, ribosome binding sites, transcription or translation. Preferably, the one or more genetic modifications include knock-in, substitution, disruption, deletion, or knockout of expression control sequences selected from the group consisting of initiation and termination sequences, enhancers, and activator sequences; including the incorporation of a heterologous polynucleotide or expression cassette into.

According to certain embodiments, the one or more genetic modifications include an increase in the number of TIF genes or expression cassettes comprising the TIF genes, and/or gain-of-function changes in the TIF genes, such that: The level or activity of the TIF gene is increased.

According to certain embodiments, the one or more genetic modifications include gain-of-function changes in the respective TIF genes, such as by overexpressing the respective TIF genes and/or by reducing degradation. or increasing the level or activity of TIF by increasing the stability of the respective TIF gene or TIF mRNA.

Specifically, the gain-of-function changes include knock-ins of the respective TIF genes.

Specifically, the gain-of-function change upregulates the expression of the respective TIF gene in the cell.

Specifically, the gain-of-function change involves the insertion of a heterologous expression cassette to overexpress the respective TIF gene in the cell.

Gain-of-function changes are specific for increasing expression of the TIF gene, e.g., by introducing a polynucleotide encoding TIF (or a TIFEC comprising such a polynucleotide) into the host cell genome, and optionally , destroying the promoter operably linked to such polynucleotide, replacing such promoter with another promoter having higher promoter activity.

Particular methods of modifying gene expression employ modulating modifications (e.g., activation, upregulation, inactivation, inhibition, or downregulation) of regulatory sequences that modulate expression of polynucleotides (genes); For example, RNA-guided ribonuclease-related sequences used in CRISPR-based methods of modifying host cells, such as promoters, ribosome binding sites, transcription initiation or termination sequences, translation initiation or termination sequences, enhancers or activator sequences. , repressor or inhibitory sequences, signal or leader sequences, in particular those which control the expression and/or secretion of the protein.

Specifically, the one or more genetic modifications to increase expression of a TIF gene include one or more genomic mutations that increase the gene or Increase the expression of some by at least 50%, 60%, 70%, 80%, 90%, or even more than 95% (e.g., by knocking in a heterologous gene or by increasing the copy number of an endogenous gene). ), including the insertion or activation of the respective gene or genomic sequence.

Specifically, the one or more genetic modifications that increase expression include genomic mutations that constitutively improve or otherwise increase the expression of one or more endogenous polynucleotides.

Specifically, the one or more genetic modifications that increase expression include genomic mutations that introduce one or more inducible or derepressible regulatory sequences that conditionally condition the expression of one or more endogenous polynucleotides. improve or otherwise increase. Such conditionally active modifications specifically target those regulatory elements and genes and are active and/or expressed depending on the cell culture conditions.

Specifically, when host cells are used in methods for producing proteins of interest (POI), expression of polynucleotides encoding the respective TIFs is increased. Specifically, upon genetic modification, the expression of the respective TIF gene is increased under host cell culture conditions while the POI is produced.

Specifically, host cells can, for example, by knock-in of one or more respective TIF genes, have a 50%, 60%, 70%, 80%, 90%, or 95% (mol/mol) to increase the amount (eg, level, activity, or concentration) of each TIF. According to a particular embodiment, the host cell is such that it contains one or more insertions of genomic sequence(s), in particular genomic sequences encoding the respective TIF, which are integrated into the host cell genome. Genetically modified. Such host cells are typically provided as knock-in strains.

According to certain embodiments, when the host cells described herein are cultured in cell culture, the total amount of each overexpressed TIF in the host cell or host cell culture is compared to the reference amount expressed or produced by the host cell without modification, or compared to the reference amount produced in the respective host cell culture, or compared to the host cell before or without such modification. , by at least 50%, 60%, 70%, 80%, 90%, or 95%, or even 100% or more (% activity or mol/mol).

According to certain embodiments, one or more of the TIF genes are endogenous or heterologous to the host cell. Specifically, the TIF genes are included in each TIF expression cassette (TIFEC). Specifically, the TIF gene is expressed in one or more TIFECs.

In particular, the TIF gene is included in one or more heterologous expression cassettes, particularly a heterologous expression construct comprising one or more expression control sequences (e.g., a promoter) operably linked to the TIF gene. include. Specifically, the expression construct is not naturally present in the host cell or is located in the genome or chromosome of the host cell at a different site than the respective endogenous TIF gene or expression construct is naturally present. integrated or provided on an episomal plasmid.

Specifically, any one, two, three, four, or five, or more, or all TIFECs contain a promoter, referred to as a TIF expression cassette (TIFEC) promoter.

Specifically, the TIFEC promoter is a constitutive promoter in which the TIFEC promoter is operably linked to the TIF gene to be expressed, or a regulatable promoter such as an inducible or derepressible promoter.

Specifically, at least one TIFEC promoter (e.g., any one or more or all TIFEC promoters used in a TIF expression cassette within the same host cell) is P. pAOX1 of K. pastoris (particularly K. pastoris or K. phaffii) or is not methanol inducible. The pAOX1 promoter is a P. is understood as the natural promoter of the "AOX1" gene, referred to as the natural gene encoding alcohol oxidase 1 of P. pastoris alcohol oxidase 1.

According to certain embodiments, the host cell further comprises an expression cassette comprising a GOI and one or more expression control sequences operably linked to the GOI for expressing the GOI in host cell culture. include.

Specifically, the GOI is expressed in a GOI expression cassette (GOIEC) that is separate from the TIF expression cassette.

Specifically, GOIEC includes a promoter referred to as the GOI Expression Cassette (GOIEC) promoter.

Specifically, the GOIEC promoter is a regulatable promoter (eg, an inducible or derepressible promoter or a constitutive promoter), and the GOIEC promoter is operably linked to the expressed GOI.

Preferably, at least one, eg any one or more or all, TIFEC promoters used in the TIF expression cassette within the same host cell are any other than the GOIEC promoter.

in particular,
a) any one or more or all TIFEC promoters are constitutive and the GOIEC promoter is an inducible or (de)repressible promoter;
b) any one or more or all TIFEC promoters are inducible or (de)repressible and the GOIEC promoter is a constitutive promoter;
c) any one or more or all of the TIFEC promoters are constitutive and the GOIEC promoter is a constitutive promoter of a different type or strength than any one or more of such TIFEC promoters;
d) Any one or more or all TIFEC promoters are inducible or (de)repressible, and the GOIEC promoter is of a different type or strength of inducibility or It is a (de)repressible promoter.

According to certain embodiments, the GOIEC promoter has increased promoter strength compared to any of the TIFEC promoters.

In a preferred embodiment, expression of the respective TIF-encoding polynucleotide is driven by a constitutive promoter and expression of the POI-encoding polynucleotide (gene) is driven by an inducible promoter. In yet another preferred embodiment, expression of the respective TIF-encoding polynucleotide is driven by an inducible promoter and expression of the POI-encoding polynucleotide (gene) is driven by a constitutive promoter.

As an example, expression of a polynucleotide encoding TIF can be driven by a constitutive GAP promoter, and expression of a polynucleotide encoding POI can be driven by a methanol-inducible promoter, such as the AOX1 or AOX2 promoter.

As another example, expression of a polynucleotide encoding TIF is driven by a constitutive promoter such as MDH3, POR1, PDC1, FBA1-1, or GPM1 (Prielhofer et al. 2017, BMC Sys Biol. 11:123). expression of the polynucleotide encoding the POI can be driven by a methanol-inducible promoter, such as the AOX1 or AOX2 promoter.

As another example, expression of a polynucleotide encoding a TIF can be driven by a constitutive promoter, such as the GAP promoter, and expression of a polynucleotide encoding a POI can be driven by a derepressed promoter such as those further described herein. can be driven by a sexual promoter.

As another example, expression of a polynucleotide encoding TIF can be driven by a constitutive promoter, and expression of a polynucleotide encoding POI can be driven by a derepressible promoter, such as those further described herein. can be done.

As another example, expression of a polynucleotide encoding TIF can be driven by a derepressible promoter, and expression of a polynucleotide encoding POI can be driven by a derepressible promoter, such as those further described herein. Can be driven.

Specifically, the expression cassettes referred to herein include at least one promoter and a polynucleotide (or gene) expressed under the transcriptional control of the promoter, and optionally a ribosome binding site, e.g. Further selected from the group consisting of transcription initiation or termination sequences, translation initiation or termination sequences, enhancer or activator sequences, repressor or inhibitory sequences, signal or leader sequences, in particular those controlling the expression and/or secretion of the protein. Contains regulatory sequences.

Specifically, expression cassettes are used that are heterologous to the host cell. In particular, the expression cassette includes a promoter operably linked to the polynucleotide, the promoter and the polynucleotide being heterologous to each other (meaning not occurring in such a combination in nature), e.g. Either one (or only one) of the nucleotides is artificial or heterologous to the other and/or the host cell described herein, or the promoter is an endogenous promoter and the polynucleotide is heterologous. or the promoter is an artificial or heterologous promoter, and the polynucleotide is an endogenous polynucleotide, and both the promoter and the polynucleotide are , artificial, xenogeneic, or derived from a different source (eg, from a different species or cell type). Specifically, the promoter is not naturally associated with and/or operably linked to the polynucleotide in the cell used as a host cell described herein.

According to certain embodiments, the heterologous expression cassette is contained in an autonomously replicating vector or plasmid, or integrated into the chromosome of the host cell.

A GOI expression construct may optionally include or consist of an expression control sequence (eg, a promoter) operably linked to the GOI to express the GOI from the expression construct in a host cell. The GOI expression construct may be included in a separate expression cassette, or an expression cassette that also expresses one or more of the TIF genes.

According to certain embodiments, the host cell is a recombinant host cell comprising at least one heterologous GOIEC, and at least one component or combination of components comprised in the GOIEC is heterologous to the host cell. In particular, artificial expression cassettes are used, in particular the promoter and the GOI are heterologous to each other, such a combination does not exist in nature, for example, one (or only one) of the promoter and the GOI can be used in combination with the other and/or the GOI. or the promoter is an endogenous promoter and the GOI is a heterologous GOI, or the promoter is an artificial or heterologous promoter to the host cells described herein. and the GOI is an endogenous GOI, and both the promoter and the GOI are artificial, xenogeneic, or from a different source (e.g., a different species or cell type) compared to the host cells described herein. Co., Ltd.). Specifically, the GOIEC promoter is not naturally associated with and/or operably linked to the GOI in the cells used as host cells described herein.

Specifically, the host cell is
a) an expression system for expressing one or more of the TIF genes in one or more heterologous TIF expression cassettes, each comprising one or more expression control sequences operably linked to the TIF gene; an expression system comprising;
b) a GOI expression cassette comprising a GOI and one or more expression control sequences operably linked to the GOI;
The expression system of a) and the expression cassette of b) are engineered to express the respective TIF gene and GOI when culturing host cells in cell culture.

According to certain embodiments, the host cell comprises an expression system that expresses one, two, three, four, or five or more of the TIF genes in one or more heterologous expression cassettes. each comprising one or more expression control sequences operably linked to the TIF gene. In certain embodiments, each TIF gene is operably linked to a TIFEC promoter.

The number of GOIECs or TIEFECs per cell typically determines the amount of each expression product. The host cells specifically contain at least one GOIEC and at least one TIFEC copy per cell. One expression cassette is typically referred to as a "single copy."

According to certain embodiments, the number of one type of GOIEC or GOIEC copies per host cell is at least (or at most) 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, or There may be even more, up to any one of 20, 30, 40, or 50.

According to certain embodiments, the number of one type of TIFEC or TIFEC copies per host cell is at least (or at most) any one of 1, 2, 3, 4, or 5.

According to certain embodiments, the number of heterologous TIFECs per host cell is at least (or at most) 1, 2, 3, 4, or 5.

TIFECs are preferably xenogeneic to the host cell, whereas GOIECs can be xenogeneic or endogenous.

According to certain embodiments, the host cell comprises one or more (e.g., a plurality) of heterologous expression cassettes, including, for example, one or more expression cassettes expressing TIF and one expressing GOI. or more copies (eg, at least 1, 2, 3, 4, or 5 copies (gene copy number, GCN) of TIFEC or GOIEC) of an expression cassette. Each copy may contain or consist of the same or different sequences, and includes expression control sequences operably linked to the respective gene to be expressed.

Specifically, for each TIF overexpressed in a host cell, the number of TIF-encoding polynucleotides per cell is approximately the same (+/-1) and at approximately the same level as for other overexpressed TIFs. Ensure all overexpression of TIF.

According to certain aspects,
a) at least one (i.e., any or all of the TIF expression cassettes) comprises a constitutive promoter, and/or b) the GOI expression cassette is inducible, derepressible, or otherwise If not, it includes a regulatable promoter or a constitutive promoter.

Specifically, GOI is a polynucleotide or gene different from the TIF gene. Specifically, GOI expresses a protein of interest (POI). Specifically, the POI is a polypeptide or protein that is different from the TIF.

According to certain embodiments, the POI is heterologous to the host cell type.

According to certain embodiments, the POI is a therapeutic or diagnostic agent. Preferably, the POI is a therapeutic protein that is functional in mammals.

Specifically, POIs include antigen binding proteins, therapeutic proteins, enzymes, peptides, protein antibiotics, toxin fusion proteins, carbohydrate-protein conjugates, structural proteins, regulatory proteins, vaccine antigens, growth factors, hormones, cytokines, A peptide, polypeptide, or protein selected from the group consisting of process enzymes and metabolic enzymes.

Specifically, the POI is a eukaryotic protein, preferably a mammalian-derived or related protein (eg, a human protein or a protein comprising a human protein sequence), or a bacterial protein or protein of bacterial origin. Any such mammalian, bacterial, or man-made protein that does not naturally occur in a yeast host cell is understood to be xenologous to the host cell.

In certain cases, the POI is a multimeric protein (particularly a dimer or a tetramer).

Specifically, the antigen binding protein is
a) Antibodies or antibody fragments (e.g. chimeric antibodies, humanized antibodies, bispecific antibodies, Fabs, Fd, scFv, diabodies, triabodies, Fv tetramers, minibodies, or single domain antibodies (VH, VHH, (such as IgNAR or V-NAR),
b) an antibody mimetic (e.g., adnectin, affibody, affilin, affimer, affitin, alphabody, anticalin, avimer, darpin, finomer, Kunitz domain peptide, monobody, or nanoclamp), or c) one or more A fusion protein comprising an immunoglobulin fold domain, an antibody domain, or an antibody mimetic.

A specific POI is an antigen-binding molecule, such as an antibody, or a fragment thereof, particularly an antibody fragment comprising an antigen-binding domain. Among the specific POIs are monoclonal antibodies (mAbs), immunoglobulins (Ig) or immunoglobulin class G (IgG), heavy chain antibodies (HcAbs), or fragments thereof (e.g., fragment antigen-binding (Fab)); Fd, single chain variable fragments (scFv), or engineered variants thereof, such as Fv dimers (diabodies), Fv trimers (triabodies), Fv tetramers, or minibodies and single domains. Antibodies include antibodies (such as VH, VHH, IgNAR, or V-NAR), or any protein that contains an immunoglobulin fold domain. Further antigen-binding molecules may be selected from antibody mimetics or (alternative) scaffold proteins such as, for example, engineered Kunitz domains, adnectins, affibodies, affilins, anticalins, or darpins.

According to certain embodiments, the POI is, for example, BOTOX, Myobloc, Neurobloc, Dysport (or other serotypes of botulinum neurotoxin), alglucosidase alpha, daptomycin, YH-16, chorionic gonadotropin alpha, filgrastim. , cetrorelix, interleukin-2, aldesleukin, teseroidin, denileukin diftitox, interferon α-n3 (injection), interferon α-nl, DL-8234, interferon, Suntory (γ-1a), interferon γ, Thymosin alpha 1, Tasonermin, DigiFab, ViperaTAb, EchiTAb, CroFab, nesiritide, abatacept, alefacept, Rebif, eptothermin alpha, teriparatide (osteoporosis), calcitonin injection (bone disease), calcitonin (nasal, osteoporosis), etanercept, hemoglobin gluta Mar 250 (bovine), drotrecogin alfa, collagenase, carperitide, recombinant human epidermal growth factor (topical gel, wound healing), DWP401, darbepoetin alfa, epoetin omega, epoetin beta, epoetin alfa, desirudin, lepirudin, bivalirudin, nonacog Alpha, mononin, eptocog alpha (activation), recombinant factor VIII + VWF, recombinant factor VIII, factor VIII (recombinant), Alphnmate, octocog alpha, factor VIII, palifermin, indi Kinase, tenecteplase, alteplase, pamiteplase, reteplase, nateplase, monteplase, follitropin alfa, rFSH, hpFSH, micafungin, pegfilgrastim, lenograstim, nartograstim, sermorelin, glucagon, exenatide, pramlintide, iniglucerase, galsulfase, Leukotropin, molgramostirn, triptorelin acetate, histrelin (subcutaneous implant, Hydron), deslorelin, histrelin, nafarelin, leuprolide sustained release depot (ATRIGEL), leuprolide implant (DUROS), goserelin, utropin, KP-102 Program, somatropin, mecasermin (growth failure), enfavirtide, Org-33408, insulin glargine, insulin glulisine, insulin (inhalation), insulin lispro, insulin deternir, insulin (buccal, RapidMist), mecasermin Linfabat, anakinra, sermolleukin, 99mTc-apsitide injection, myelopid, betaserone, glatiramer acetate, gepon, sargramostim, oprelvekin, human leukocyte-derived alpha interferon, Vilib, insulin (recombinant), recombinant human insulin, insulin aspart , mecasenin, Roferon-A, Interferon-α2, Alphaferon, Interferon Alphacon-1, Interferon-α, Avonex Recombinant Human Luteinizing Hormone, Dornase Alpha, Trafermin, Ziconotide, Taltireline, Dibotermin Alpha, Atosiban , becapermin, eptifibatide, Zemaila, CTC-111, Chambac-B, HPV vaccine (quadrivalent), octreotide, lanreotide, ancestirn, agalsidase beta, agalsidase alpha, laronidase, prezatide copper acetate (topical gel), Rasburicase, ranibizumab, actimmune, pegintron, trichomine, recombinant house dust mite allergy desensitization injection, recombinant human parathyroid hormone (PTH) 1-84 (subcutaneous, osteoporosis), epoetin delta, transgenic antithrombin III, granditropin , vitrase, recombinant insulin, interferon alpha (oral lozenge), GEM-21S, vapreotide, idursulfase, omnapatrilat, recombinant serum albumin, certolizumab pegol, glucarpidase, human recombinant C1 esterase inhibition drug (angioedema), lanoteplase, recombinant human growth hormone, enfuvirtide (needle-free injection, Biojector 2000), VGV-1, interferon (α), lucinactant, aviptadil (inhalation, pulmonary disease), icatibant, ecalantide, omiganan, auroglob , pexiganan acetate, ADI-PEG-20, LDI-200, degarelix, sintredequin vesdotox, FavId, MDX-1379, ISAtx-247, liraglutide, teriparatide (osteoporosis), tifacogin, AA4500, T4N5 liposome lotion, katumaxomab, DWP413, ART-123, Chrysaline, Desmoteplase, Amediplase, Corifollitropin alpha, TH-9507, Teduglutide, Diamide, DWP-412, Growth hormone (sustained release injection), Recombinant G-CSF, Insulin (inhalation, AIR) ), insulin (inhalation, Technosphere), insulin (inhalation, AERx), RGN-303, DiaPep277, interferon beta (hepatitis C virus infection (HCV)), interferon alpha-n3 (oral), belatacept, transdermal insulin patch, AMG-531, MBP-8298, Xerecept, Opebacan, AIDSVAX, GV-1001, Lymphoscan, Lanpirnase, Lipoxisan, Lusupletide, MP52 (tricalcium phosphate carrier, bone regeneration), Melanoma vaccine, Sipuleucel-T, CTP-37 , Inthesia, Vitespen, human thrombin (freezing, surgical bleeding), thrombin, TransMID, alfimeplase, pulicase, terlipressin (intravenous, hepatorenal syndrome), EUR-1008M, recombinant FGF-I (injection, vascular disease), BDM-E, rotigaptide, ETC-216, P-113, MBI-594AN, duramycin (inhalation, cystic fibrosis), SCV-07, OPI-45, endostatin, angiostatin, ABT-510, Bowman Burke Inhibitor Concentrate, XMP-629, 99mTc-Hynic-Annexin V, Kahalalide F, CTCE-9908, Teverelix (extended release), Ozarelix, ronidepsin, BAY-504798, Interleukin-4, PRX-321, Pepscan, Ivotadequin , rh lactoferrin, TRU-015, IL-21, ATN-161, cilengitide, albuferon, Biphasix, IRX-2, omega interferon, PCK-3145, CAP-232, pasireotide, huN901-DMI, ovarian cancer Immunotherapy vaccines, SB-249553, Oncovax-CL, OncoVax-P, BLP-25, CerVax-16, Multi-epitope peptide melanoma vaccine (MART-1, gp100, tyrosinase), Nemifitide, rAAT (inhalation), rAAT (dermal) ), CGRP (inhalation, asthma), pegsnercept, thymosin beta 4, plitidepsin, GTP-200, ramoplanin, GRASPA, OBI-1, AC-100, salmon calcitonin (oral, eligen), calcitonin (oral, osteoporosis), examorelin, Capromorelin, Cardeva, bellafermin, 131I-TM-601, KK-220, T-10, uralitide, deperestat, hematide, Chrysalin (topical), rNAPc2, recombinant factor V111 (PEGylated liposome), bFGF, PEGylated recombinant Staffylokinaze Variant, V -10153, SONOLYSIS PROLYSE, NEUROVAX, CZEN -002, pancreatic island cell new therapy, RGLP -1, Bim -51077, LY -548806, eccenatide (release control, MEDISORB), AVE -0010, GA -GCB , aborelin, ACM-9604, linaclotide acetate, CETi-1, Hemospan, VAL (injection), rapid-acting insulin (injection, Viadel), intranasal insulin, insulin (inhalation), insulin (oral, eligen), recombinant Methionylhuman leptin, Pitrakinra subcutaneous injection, eczema), Pitrakinra (inhalation dry powder, asthma), Multicaine, RG-1068, MM-093, NBI-6024, AT-001, PI-0824, Org-39141, Cpn10 ( autoimmune disease/inflammation), talactoferrin (topical), rEV-131 (ophthalmology), rEV-131 (respiratory disease), oral recombinant human insulin (diabetes), RPI-78M, oprelvekin (oral), CYT-99007 , CTLA4-Ig, DTY-001, Balategrast, Interferon alpha-n3 (topical), IRX-3, RDP-58, Tauferon, Bile salt-stimulated lipase, Merispase, Alaline phosphatase, EP-2104R, Melanotan-II , bremelanotide, ATL-104, recombinant human microplasmin, AX-200, SEMAX, ACV-1, Xen-2174, CJC-1008, dynorphin A, SI-6603, LAB GHRH, AER-002, BGC-728, Malaria vaccine (Virosome, PeviPRO), ALTU-135, parvovirus B19 vaccine, influenza vaccine (recombinant neuraminidase), malaria/HBV vaccine, anthrax vaccine, Vacc-5q, Vacc-4x, HIV vaccine (oral), HPV vaccine , Tat toxoid, YSPSL, CHS-13340, PTH (1-34) liposome cream (Novasome), Ostabolin-C, PTH analog (topical, psoriasis), MBRI-93.02, MTB72F vaccine (tuberculosis), MVA-Ag85A Vaccine (tuberculosis), FARA04, BA-210, recombinant plague FIV vaccine, AG-702, OxSODrol, rBetV1, Der-p1/Der-p2/Der-p7 allergen targeting vaccine (house dust mite allergy), PR1 peptide antigen ( leukemia), mutant ras vaccine, HPV-16E7 lipopeptide vaccine, labyrinthine vaccine (adenocarcinoma), CML vaccine, WT1-peptide vaccine (cancer), IDD-5, CDX-110, Pentrys, Norelin, CytoFab, P -9808, VT-111, iclocaptide, terbermine (dermatology, diabetic foot ulcer), lupintrivir, reticulose, rGRF, HA, α-galactosidase A, ACE-011, ALTU-140, CGX-1160, angiotensin treatment vaccine, D-4F, ETC-642, APP-018, rhMBL, SCV-07 (oral, tuberculosis), DRF-7295, ABT-828, ErbB2-specific immunotoxin (anticancer drug), DT3SSIL-3, TST-10088, PRO-1762, Combotox, cholecystokinin B/gastrin receptor binding peptide, 111In-hEGF, AE-37, trasnizumab-DM1, antagonist G, IL-12 (recombinant), PM-02734 , IMP-321, rhIGF-BP3, BLX-883, CUV-1647 (topical), L-19-based radioimmunotherapy (cancer), Re-188-P-2045, AMG-386, DC/1540/ KLH vaccine (cancer), VX-001, AVE-9633, AC-9301, NY-ESO-1 vaccine (peptide), NA17. A2 peptide, melanoma vaccine (pulse antigen therapy), prostate cancer vaccine, CBP-501, recombinant human lactoferrin (dry eye), FX-06, AP-214, WAP-8294A (injection), ACP-HIP , SUN-11031, Peptide YY[3-36] (obesity, intranasal), FGLL, atacicept, BR3-Fc, BN-003, BA-058, human parathyroid hormone 1-34 (nasal, osteoporosis), F- 18-CCR1, AT-1100 (celiac disease/diabetes), JPD-003, PTH (7-34) liposome cream (Novasome), duramycin (ophthalmology, dry eye), CAB-2, CTCE-0214,

GlycoPEGylated erythropoietin, EPO-Fc, CNTO-528, AMG-114, JR-013, Factor immediate release), EP-51216, hGH (controlled release, Biosphere), OGP-I, sifuvirtide, TV4710, ALG-889, Org-41259, rhCC10, F-991, thymopentin (lung disease), r(m)CRP, Liver-selective insulin, subarin, L19-IL-2 fusion protein, elafin, NMK-150, ALTU-139, EN-122004, rhTPO, thrombopoietin receptor agonist (thrombocytopenic disease), AL-108, AL-208 , nerve growth factor antagonist (pain), SLV-317, CGX-1007, INNO-105, oral teriparatide (eligen), GEM-OS1, AC-162352, PRX-302, LFn-p24 fusion vaccine (Therapore), EP -1043, pneumococcal pediatric vaccine, malaria vaccine, meningococcal group B vaccine, neonatal group B streptococcal vaccine, anthrax vaccine, HCV vaccine (gpE1+gpE2+MF-59), otitis media therapy, HCV vaccine (core antigen + ISCOMATRIX), hPTH ( 1-34) (dermal, ViaDerm), 768974, SYN-101, PGN-0052, Aviscumin, BIM-23190, tuberculosis vaccine, multi-epitope tyrosinase peptide, cancer vaccine, encastim, APC-8024, GI-5005, ACC -001, TTS-CD3, Vascular Targeting TNF (Solid Tumors), Desmopressin (Buccal Controlled Release), Onercept or TP-9201, Adalimumab (HUMIRA), Infliximab (REMICADE™), Rituximab (RITUXAN™/ MAB THERA™), etanercept (ENBREL™), bevacizumab (AVASTIN™), trastuzumab (HERCEPTIN™), pegrilgrastim (NEULASTA™), or biosimilars and Any other suitable POI including Biobetter.

Specifically, the POI is heterologous to the host cell type.

Specifically, a POI is a secreted peptide, polypeptide, or protein, ie, secreted from a host cell into the cell culture supernatant.

Specifically, the GOI is expressed with a secretory signal sequence, preferably a secretory signal peptide (or a leader comprising a secretory signal peptide) fused to the N-terminus of the POI.

The invention further provides methods for producing the host cells described herein. Specifically, such methods involve genetically engineering a host cell to include one or more of the TIF genes and a gene of interest (GOI) in one or more heterologous expression cassettes.

According to a further specific aspect, the invention provides for genetically engineering a host cell to operably link two or more heterologous nucleic acid molecules, and each of the heterologous nucleic acid molecules, into one or more expression cassettes. Provided are methods for producing a host cell as described herein that is capable of producing a protein of interest (POI) in host cell culture by introducing an expression control sequence in which one of the nucleic acid molecules One comprises a gene of interest (GOI) encoding a POI, and the additional one or more nucleic acid molecules encode a TIF, such as a TIF gene, as further described herein.

Specifically, host cells are provided by genetic engineering of wild-type host cells.

According to a specific example, a host cell can be produced by first modifying it to introduce one or more expression cassettes that express the TIF gene of interest. Such modified host cells can then be further engineered to contain expression cassettes for POI production.

According to another specific example, a host cell can be produced by a first operation to contain an expression cassette for POI production. Such engineered host cells can be further modified to introduce one or more expression cassettes that express the TIF gene.

The present invention is a method for producing a protein of interest (POI) encoded by a gene of interest (GOI), wherein the production is performed under the conditions described herein for producing the POI. Further provided is a method carried out by culturing a host cell of.

The invention further provides a method for producing a protein of interest (POI) in a host cell,
(i) genetically engineering a host cell to include the TIF gene and a gene of interest (GOI) encoding the POI in one or more heterologous expression cassettes as described herein; ,
(ii) culturing the host cell in a culture medium under conditions that co-express the TIF gene and the GOI, thereby obtaining a POI;
(iii) recovering the POI from the host cell or culture medium.

In particular, step i) of the method described herein is performed before step (ii).

According to a particular example, a wild type host cell is genetically modified according to step i) of the method described herein.

Specifically, a host cell is provided for introducing the genetic modification into a wild-type host cell line for expressing a heterologous expression cassette. However, according to certain embodiments, the host cell is modified, e.g., prior to genetic modification according to step i), in order to improve the cell's ability to express and/or secrete proteins, or to produce unwanted by-products, e.g. The wild-type host cell may undergo one or more further genetic modifications to reduce host cell proteins).

Specifically, suitable method steps are used to produce recombinant host cells as further described herein.

Specifically, the POI is obtained by culturing host cells in a suitable medium, isolating the expressed POI from the cell culture (particularly from the cell culture supernatant or medium when separating the cells), and culturing the expressed POI in a suitable medium. The POI can be produced by purifying it by any suitable method, particularly when separating the POI from the cells, by any suitable means. This may produce a purified POI preparation.

In particular, the methods described herein are characterized by the features further described herein (in particular by the recombinant host cells and/or expression systems further described herein).

According to certain aspects, the invention further provides the use of a host cell described herein for the production of POI.

Specifically, POIs are produced by expressing the GOI while culturing host cells under conditions that co-express or overexpress one or more of the TIF genes. Specifically, such methods increase the expression of the GOI and the production yield of the POI.

Specifically, host cells are cultured in a culture medium under conditions to co-express one or more of the TIF genes and secrete the POI into the host cell culture, and the POI is added to the host cell culture. recovered from objects.

Specifically, the host cell is a cell line (particularly a production host cell line) grown in cell culture.

According to certain embodiments, cell lines are cultured under suitable batch, fed-batch, or continuous culture conditions. Cultivation may be carried out in microtiter plates, shake flasks, or bioreactors, optionally starting with a batch phase as a first step, followed by a fed-batch phase or continuous cultivation as a second step. Phases may continue.

Specifically, the cell culture employs growing cells in a batch phase and culturing cells to produce the POI in a fed-batch or continuous culture phase, optionally comprising: Starting with a batch phase as a first step, followed by a fed-batch or continuous culture phase as a second step.

According to certain embodiments, the methods described herein include a growth phase and a production phase.

Specifically, the method:
a) culturing a host cell under growth conditions (a growing phase or "growth phase"); and b) culturing a host cell in which a GOI is expressed to produce the POI. and culturing under growth-limiting conditions (production phase).

In particular, the second step b) follows the first step a).

Specifically, the host cell is at a level that increases the specific productivity (μg/g yeast dry mass (YDM)/hour) and/or volumetric productivity (μg/L/hour) of the host cell for the POI. and are modified to co-express one or more of the TIF genes.

Specifically, such co-expression results in a 1.2-fold, 1.3-fold, 1.4-fold increase in the TIF compared to an equivalent host cell expressing the GOI that has not been engineered to co-express the TIF. times, 1.5 times, 1.6 times, 1.7 times, 1.8 times, 1.9 times, 2.0 times, 2.1 times, 2.2 times, 2.3 times, 2.4 times, 2.5 times, 2.6 times, 2.7 times, 2.8 times, 2.9 times, 3 times, 3.5 times, 4 times, 5 times, 5.5 times, 6 times, 6 .5 times, 7 times, 7.5 times, 8 times, 8.5 times, 9 times, 9.5 times, 10 times, 10.5 times, 11 times, 11.5 times, or 12 times. Productivity or yield increases in at least one of them.

When comparing the host cells described herein for the effect of genetic modification to produce the TIF, a comparison is typically made to an equivalent host cell before or without such genetic modification. Comparisons are typically made to the same host cell type or type without such genetic modification that is engineered to produce the POI, particularly when cultured under conditions in which the POI is produced. However, comparisons can also be made to the same host cell type or type that has not been further engineered to produce POI. The production of the TIF upon expression of the respective coding sequence can be determined by the amount (eg, level or concentration) of the TIF produced by the host cell. Specifically, the amount can be determined by Western blot, immunofluorescence imaging, flow cytometry, or mass spectrometry (in particular, mass spectrometry can be performed using liquid chromatography-mass spectrometry (LC-MS) or liquid chromatography-tandem mass spectrometry (LC-MS)). MS/MS).

According to certain embodiments, the host cells described herein may undergo one or more additional genetic modifications, eg, to improve protein production.

Specifically, the host cell is further engineered to modify one or more genes affecting proteolytic activity used to generate a protease-deficient strain, in particular a strain deficient in carboxypeptidase Y activity. Specific examples are described in WO 1992/017595A1. Further examples of protease-deficient Pichia strains having a functional defect in vacuolar proteases such as protease A or protease B are described in US6153424A. Further examples are Pichia strains with deletions of ade2 and/or deletions of one or both of the protease genes PEP4 and PRB1, such as those provided by ThermoFisher Scientific.

Specifically, the host cell is selected from the group consisting of PEP4, PRB1, YPS1, YPS2, YMP1, YMP2, YMP1, DAP2, GRHI, PRD1, YSP3, and PRB3, as disclosed in WO2010/099195A1. can be engineered to modify at least one nucleic acid sequence encoding a functional gene product (particularly a protease).

Overexpression or underexpression of genes encoding helper factors can be applied to enhance the expression of GOIs, for example as described in WO2015/158800A1.

According to certain embodiments, the host cell is a eukaryotic host cell.

Specifically, the host cell is
a) Yeast cells of a genus selected from the group consisting of Pichia, Hansenula, Komagataella, Saccharomyces, Krivellomyces, Candida, Ogataea, Yarrowia, and Geotrichum (preferably Pichia pastoris, Komagataella phaffii, Komagataella pastoris, Komagataella pseudopastoris, Saccharomyces cerevisiae, Ogataea minuta, Kluyveromces lactis, Kluyveromes marxianus, Yarrowia lipolytica, or Hansenula polymorpha), or b) cells of filamentous fungi (e.g., Aspergillus awamori or Trichoderma reesei), or c) non-human primates. , human, rodent, or bovine cells (e.g., mouse myeloma (NSO) cell line, Chinese hamster ovary (CHO) cell line, HT1080, H9, HepG2, MCF7, MDBK, Jurkat, MDCK, NIH3T3, PC12, BHK (baby hamster kidney cells), VERO, SP2/0, YB2/0, Y0, C127, L cells, COS (COS1 and COS7, etc.), QC1-3, HEK-293, VERO, PER.C6, HeLA, EBI, EB2, EB3, oncolytic or hybridoma cell lines), or d) insect cells (e.g. Sf9, Mimic™ Sf9, Sf21, High Five (BT1-TN-5B1-4), or BT1-Ea88 cells), or e) an algal cell (e.g., Amphora, Viola, Dunaliella, Chlorella, Chlamydomonas, Cyanobacteria, Nannochloropsis, Spirulina, or Ochromonas), or f) a plant cell ( For example, cells derived from monocotyledonous plants (preferably corn, rice, wheat, or hackberry) or cells derived from dicot plants (preferably cassava, potato, soybean, tomato, tobacco, alfalfa, Physcomitrella patens, or Arabidopsis). cells).

According to certain embodiments, the host cell can be any yeast cell. In particular, the host cell is a cell of a genus selected from the group consisting of Pichia, Hansenula, Komagataella, Saccharomyces, Krivellomyces, Candida, Ogataea, Yarrowia, and Geotrichum; are Saccharomyces cerevisiae, Pichia pastoris, Ogataea minuta, Kluyveromces lactis, Kluyveromes marxianus, Yarrowia lipolytica o r Hansenula polymorpha, or cells of filamentous fungi such as Aspergillus awamori or Trichoderma reesei. Preferably, the host cell is a methylvegetative yeast, preferably Pichia pastoris. As used herein, Pichia pastoris is all used synonymously with Komagataella pastoris, Komagataella phaffii, and Komagataella pseudopastoris.

Specific examples include Pichia (e.g., Pichia pastoris, Pichia methanolica, Pichia kluyveri, and Pichia angusta), Komagataella (e.g., Komagataella pastoris, Komagataell a pseudopastoris, or Komagataella phaffii), Saccharomyces (e.g., Saccharomyces cerevisae, Saccharomyces Kluyveri, Saccharomyces varum), Kluyveromyces (e.g. Kluyveromyces lactis, Kluyveromyces marxianus), Candida (e.g. Candida utilis, Candida cacaoi) , Candida boidinii), Geotrichum fermentans, Hansenula polymorpha, Yarrowia lipolytica, or Schizosaccharomyces pombe yeast cells.

The species Pichia pastoris is preferred. Specifically, the host cell is a Pichia pastoris strain selected from the group consisting of CBS704, CBS2612, CBS7435, CBS9173-9189, DSMZ 70877, X-33, GS115, KM71, KM71H, and SMD1168.

Source: CBS704 (=NRRL Y-1603=DSMZ70382), CBS2612 (=NRRL Y-7556), CBS7435 (=NRRL Y-11430), CBS9173-9189 (CBS stock: CBS-KNAW Fungal Biodive rsity Centre, Centralbureau voor Schimmelculturen, Utrecht , The Netherlands), and DSMZ 70877 (German Collection of Microorganisms and Cell Cultures); Strain from ermo Fisher.

Preferred S. Examples of S. cerevisiae strains include W303, CEN. PK, and BY series (EUROSCARF collection). All of the above strains have been successfully used to produce transformants and express heterologous genes.

The present invention provides a method for increasing the yield of a protein of interest (POI), comprising messenger ribonucleoprotein in cell culture when produced by a host cell expressing a gene of interest (GOI) encoding the POI. Further provided are methods for increasing the expression of one or more TIF genes (mRNP) by co-expressing one or more heterologous expression cassettes.

The present invention further provides a polypeptide expression system comprising one or more heterologous expression cassettes expressing one or more TIF genes of a messenger ribonucleoprotein (mRNP), such as the TIF genes further described herein. . Such expression cassettes are also referred to herein as TIF expression constructs or TIF (TIF gene) expression cassettes (TIFEC). Specifically, the heterologous expression cassette includes one or more expression control sequences operably linked to the TIF gene for expressing the TIF gene, particularly, such as, for example, a promoter, a signal peptide, or a leader. at least one of the expression control sequences of the TIF gene is not naturally operably linked to the TIF gene. Specifically, the TIF expression cassette is characterized as further described herein.

Specifically, the expression system further includes an expression cassette that includes a GOI encoding a protein of interest (POI) and one or more expression control sequences operably linked to the GOI. Such expression cassettes are also referred to herein as GOI expression constructs (GOIECs) or GOI expression cassettes. Specifically, the GOI expression cassette is characterized as further described herein. Specifically, the expression cassette containing the GOI is separate from other expression cassettes expressing the TIF gene.

In particular, the expression systems described herein are characterized by the expression cassette and recombinant expression construct features further described herein.

Specifically, the TIF genes overexpressed by the genetic manipulation are each contained in separate expression cassettes. However, expression cassettes containing at least two or three TIF genes and optionally further containing a GOI may also be used.

The present invention provides host cells (particularly host cells as described herein) comprising an expression system described herein (particularly an expression system comprising an expression cassette expressing a TIF gene and an expression cassette expressing a GOI). cells) are further provided.

According to a particular embodiment, the host cell is a recombinant host cell comprising at least one heterologous GOIEC comprising an expression cassette promoter operably linked to the GOI; The combination is heterologous to the host cell.

In particular, artificial expression cassettes are used, in particular where the promoter and the gene expressed under the control of the promoter are heterologous to each other (such a combination does not exist in nature), e.g. or only one) is artificial or heterologous to the other and/or the host cells described herein, the promoter is an endogenous promoter, and the gene expressed is a heterologous gene , or the promoter is an artificial or heterologous promoter, and the gene is an endogenous gene, and both the promoter and the gene are artificial, heterologous, or be from a different source (eg, from a different species or cell type).

According to certain embodiments, any one or more (or all) of the heterologous expression cassettes are contained in one or more autonomously replicating vectors or plasmids, or integrated into the chromosome of the host cell. It will be done.

The expression cassette may be introduced into the host cell and integrated into the host cell genome (or any of its chromosomes) as an intrachromosomal element, e.g., at a specific site of integration or randomly, and then integrated into the host cell genome (or any of its chromosomes), e.g., at a specific site of integration or randomly. Stocks are selected. Alternatively, the expression cassette may be integrated within an extrachromosomal genetic element such as a plasmid or an artificial chromosome, eg, a yeast artificial chromosome (YAC). According to a particular example, the expression cassette is introduced into the host cell by a vector (particularly an expression vector) that is introduced into the host cell by suitable transformation or transfection techniques. For this purpose, the heterologous polynucleotide to be expressed (particularly the GOI) can be ligated into an expression vector.

Preferred yeast expression vectors are selected from the group consisting of plasmids derived from pPICZ, pGAPZ, pPIC9, pPICZalfa, pGAPZalfa, pPIC9K, pGAPHis, pPUZZLE, or GoldenPiCS.

Techniques for transfecting or transforming host cells to introduce vectors or plasmids are well known in the art. These include electroporation, spheroplasts, lipid vesicle-mediated uptake, heat shock-mediated uptake, calcium phosphate-mediated transfection (calcium phosphate/DNA co-precipitation), viral infections and especially modified viruses (e.g. modified adenoviruses). ), microinjection, as well as electroporation.

As used herein, the term "transforming" a yeast cell is understood to include "transfecting" it.

Transformants described herein can be obtained by introducing an expression cassette, vector, or plasmid DNA into a host and selecting transformants that express the relevant protein or selection marker. Host cells can be treated to introduce heterologous or foreign DNA by methods conventionally used for transformation of host cells, such as electrical pulse methods, protoplast methods, lithium acetate methods, and modifications thereof. Preferred transformation methods for uptake of recombinant DNA fragments by microorganisms include chemical transformation, electroporation, or transformation by protoplastization.

According to certain embodiments, expression cassettes comprising or consisting of an artificial fusion of polynucleotides are used, the artificial fusion comprising a promoter operably linked to the heterologous polynucleotide, and optionally a signal. sequence, leader sequence, or terminator sequence.

Specifically, TIFEC expresses the TIF as an intracellular protein.

Specifically, GOIEC contains the signal and leader sequences necessary to express and produce POI as a secreted protein.

According to a particular embodiment, the GOI is fused at the 5' end of a nucleotide sequence encoding a secretion signal sequence, preferably a heterologous secretion signal sequence.

According to certain embodiments, the GOIEC includes a nucleotide sequence encoding a signal peptide that enables secretion of the POI. Specifically, a nucleotide sequence encoding a signal peptide is fused adjacent to the 5' end of the GOI.

The signal sequence may be a natural signal sequence, and as used herein is a sequence that is coexpressed, fused, or otherwise coexpressed with a naturally occurring protein in order to secrete such a protein upon expression. It is understood as a signal sequence that is associated with For example, a natural secretory signal sequence is typically a signal sequence that is coexpressed, fused, or otherwise associated with the respective protein to be secreted. Specifically, a natural secretion signal sequence is used that is heterologous (or not naturally associated) with the POI, such as a signal sequence derived from a secreted protein different from the POI.

Alternatively, artificial secretion signal sequences can be used, particularly those that have at least one of 85%, 90%, or 95% sequence identity with those that occur in nature. .

Specifically, the signal sequence is S. The signal sequence from the alpha mating factor prepropeptide of P. cerevisiae. a signal sequence derived from the acid phosphatase gene (PHO1) of P. pastoris, and extracellular protein X (EPX1) (Heiss, S., V. Puxbaum, C. Gruber, F. Altmann, D. Mattanovich & B. Gasser, Microbiology 2015;161(7):1356-68).

Specifically, any of the signal sequences and/or leader sequences described in WO2014/067926A1 or WO2012/152823 A1 can be used.

Sequences referred to herein Sequences referred to herein Sequences referred to herein Sequences referred to herein Sequences referred to herein Sequences referred to herein Sequences referred to herein Sequences referred to herein Sequences referred to herein Sequences referred to herein Sequences referred to herein Sequences referred to herein Sequences referred to herein Sequences referred to herein Sequences referred to herein Sequences referred to herein Sequences referred to herein Sequences referred to herein Sequences referred to herein Sequences referred to herein Sequences referred to herein Sequences referred to herein Sequences referred to herein Sequences referred to herein Sequences referred to herein Sequences referred to herein Sequences referred to herein Sequences referred to herein Sequences referred to herein Sequences referred to herein Sequences referred to herein Sequences referred to herein Sequences referred to herein Sequences referred to herein Sequences referred to herein Sequences referred to herein Sequences referred to herein Sequences referred to herein Sequences referred to herein

Unless otherwise indicated or defined, all terms used herein have their ordinary meanings in the art, as would be apparent to one of ordinary skill in the art. For example, Sambrook et al, "Molecular Cloning: A Laboratory Manual" (2nd Ed.), Vols. 1-3, Cold Spring Harbor Laboratory Press (1989), Lewin, "Genes IV", Oxford University Press, New York, (1990), and Janeway et al. , "Immunobiology" (5th Ed., or more recent editions), Garland Science, New York, 2001.

As used herein, the terms "comprise," "contain," "have," and "include" may be used interchangeably. , shall be understood as an open definition allowing further members or parts or elements. "Consisting" is considered the narrowest definition that does not include further elements of the constituent defining feature. Thus, "comprising" is broader and includes the definition "consisting."

The term "about" as used herein refers to a value that is the same or different from a given value by +/-10% or +/-5%.

Certain terms used throughout this specification have the following meanings.

As used herein, the term "cell" refers to a single cell, single cell clone, or cell line of a host cell.

The term "cell line" as used herein refers to an established clone of a particular cell type that has acquired the ability to proliferate over an extended period of time. Cell lines are typically used to express endogenous or recombinant nucleic acid molecules or genes or products of metabolic pathways to produce polypeptides or cellular metabolites mediated by such polypeptides. Ru. A "production host cell line" or "production cell line" is generally understood to be a cell line prepared for cell culture in a bioreactor to obtain the product of a production process, such as a POI.

Specific embodiments described herein include at least two different polynucleotides (nucleic acid molecules or genes), at least one TIF gene encoding a TIF described herein, and at least one TIF gene encoding a POI. Refers to a production host cell line that has been engineered to co-express one gene of interest (GOI) and, in particular, the POI is produced in high yield by co-expressing the respective polynucleotides.

Host cells that produce the POIs described herein are also referred to as "production host cells," and each cell line is referred to as a "production cell line." Specific embodiments described herein refer to such POI-producing host cell lines that are engineered to co-express such TIFs and are characterized by high POI production rates.

The term "host cell" as used herein applies in particular to any cell suitably used for recombinant purposes to produce POIs or host cell metabolites. It is well understood that the term "host cell" does not include humans. Specifically, the recombinant host cells described herein are engineered organisms and derivatives of natural (wild type) host cells. The host cells, methods, and uses described herein specifically refer to those containing one or more genetic modifications, heterologous expression cassettes, or artificial expression constructs, such as transfected or transformed host cells. It is well understood that , and recombinant proteins are not naturally occurring, are "artificial" or synthetic, and are therefore not considered a result of "natural laws." The genetic modifications described herein can be performed using tools, methods, and techniques known in the art, see, for example, J. Sambrook et al. , Molecular Cloning: A Laboratory Manual (3rd edition), Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, New York (2001).

As used herein with reference to host cells, the term "cell culture" or "cultivating" or "culturing" refers to the cultivation of cells in an active or quiescent state, specifically by methods known in the art. Refers to the maintenance of cells in an artificial (e.g., in vitro) environment under conditions that favor cell proliferation, differentiation, or continued survival within a controlled bioreactor.

When culturing a cell culture using a suitable culture medium, the cells are contacted with the medium or substrate in the culture vessel under conditions suitable to support the cultivation of the cells in the cell culture. Standard cell culture media and techniques are well known in the art.

The cell culture described herein is a technique that provides for the production of secreted POI, such as to obtain POI in a cell culture medium that is separable from cell biomass, referred to herein as "cell culture supernatant." can be specifically used and purified to obtain POI with higher purity. When a protein (e.g., POI) is produced and secreted by a host cell in cell culture, such protein is secreted into the cell culture supernatant, separating the cell culture supernatant from the host cell biomass, and optionally separating the protein from the host cell biomass. It is understood that the protein can be obtained by further purification to produce a purified protein preparation.

Cell culture media provide the necessary nutrients to maintain and grow cells in a controlled, artificial in vitro environment. The characteristics and composition of cell culture media will vary depending on the specific cell requirements. Important parameters include osmolarity, pH, and nutrient composition. Supply of nutrients can be carried out in continuous or discontinuous mode according to methods known in the art.

Batch process is a cell culture mode in which all the nutrients required for the culture of cells are contained in the initial culture medium, after a batch phase, in a fed-batch or continuous process, without additional supply of further nutrients during fermentation. In the feeding phase, one or more nutrients are supplied to the culture by feeding. Although in most processes the mode of feeding is critical and important, the host cells and methods described herein are not limited with respect to the particular mode of cell culture.

Recombinant POI can be prepared by culturing the host cells and the respective cell lines described herein in a suitable medium, isolating the expressed product or metabolite from the culture, and optionally Alternatively, it can be produced by purifying it by suitable methods.

Several different approaches are preferred for producing the POIs described herein. POI involves transforming or transfecting host cells with expression vectors carrying recombinant DNA encoding the relevant proteins, preparing cultures of transformed or transfected cells, and growing cultures. inducing transcription and POI production, and recovering the POI, it can be expressed, processed, and optionally secreted.

In certain embodiments, the cell culture process is a fed-batch process. Specifically, host cells transfected with a nucleic acid construct encoding a desired recombinant POI are cultured in a growth phase and transferred to a production phase to produce the desired recombinant POI.

In another embodiment, the host cells described herein are cultured in continuous mode (eg, using a chemostat). Continuous fermentation processes are characterized by a defined, constant and continuous feed rate of fresh culture medium into the bioreactor, whereby the culture broth is also simultaneously filled with biomass at the same defined, constant and continuous removal rate. removed from the reactor. By maintaining the medium, feed rate, and removal rate at the same constant level, the cell culture parameters and conditions within the bioreactor remain constant.

In another embodiment, the host cells described herein are cultured in a perfusion mode, eg, culturing the cells within the device while providing fresh medium and removing the supernatant.

The stable cell cultures described herein maintain their genetic properties, specifically at high POI production levels, e.g., at least μg levels, even after about 20 generations of culture, preferably at least 30 It is particularly understood to refer to a cell culture that has been maintained even after generations, more preferably at least 40 generations, most preferably at least 50 generations. Specifically, stable recombinant host cell lines are provided, which is considered a major advantage when used for industrial scale production.

The cell cultures described herein are suitable for both volume and technical systems, e.g. in combination with the supply of nutrients, in particular culture modes based on fed-batch or batch processes, or continuous or semi-continuous processes (e.g. chemostats). , which is particularly advantageous for industrial manufacturing scale processes.

The host cells described herein are typically tested for their ability to express a GOI for POI production using the following tests: ELISA, activity assay, capillary electrophoresis, HPLC, or other suitable POI yield is tested by any of the following tests (eg, SDS-PAGE and Western blot techniques, or mass spectrometry).

To determine the effect of co-expressing one or more TIFs (e.g., effects on POI production), host cell lines are compared to strains that do not have such genetic modifications for co-expression in the respective cells. It can be cultured in microtiter plates, shake flasks, or bioreactors using fed-batch or chemostatic fermentation.

The production methods described herein particularly allow fermentation on a pilot scale or on an industrial scale. The industrial process scale preferably employs a volume of at least 10 L, in particular at least 50 L, preferably at least 1 m ³ , preferably at least 10 m ³ and most preferably at least 100 m ³ .

Production conditions on an industrial scale are preferred, which refers, for example, to fed-batch cultures in reactor volumes of 100 L to 10 m ³ or more, with typical process times of several days, or with dilution rates of about 0.001 Refers to a continuous process in a fermenter volume of about 50-1000 L or more, with a time period of ~0.15 h ^-1 .

The devices, equipment, and methods used for the purposes described herein are particularly suitable for use in and with the cultivation of any desired cell line. Additionally, the devices, equipment, and methods are suitable for culturing any yeast host cell type, including polypeptide products (POI), nucleic acid products (e.g., DNA or RNA), or cells and/or viruses (e.g., cells). It is particularly suitable for production operations configured for the production of pharmaceuticals and biopharmaceuticals, such as those used in therapy and/or virotherapy.

In certain embodiments, the cell expresses or produces a product, such as a recombinant therapeutic or diagnostic agent. As described in more detail herein, examples of products produced by cells include antibody molecules (e.g., monoclonal antibodies, bispecific antibodies), antibody mimetics (e.g., DARPins, affibibodies, adnectins). or polypeptide molecules that specifically bind to the antigen, such as IgNAR, but are structurally unrelated), fusion proteins (e.g., Fc fusion proteins, chimeric cytokines), other recombinant proteins (e.g., glycosylated proteins, enzymes, hormones), or viral therapeutics (e.g., anticancer oncolytic viruses, vectors for gene therapy and viral immunotherapy, and viral vectors for virotherapy), cell therapeutics (e.g., pluripotent stem cells, mesenchymal stem cells, and adult stem cells), or vaccines or lipid-encapsulated particles (eg, exosomes, virus-like particles), RNA (eg, siRNA) or DNA (eg, plasmid DNA), antibiotics, or amino acids. In embodiments, the devices, equipment, and methods can be used to produce biosimilars.

As mentioned, in certain embodiments, the devices, equipment, and methods contain eukaryotic cells (e.g., yeast cells) and/or products of the cells (e.g., proteins) synthesized by the cells in a large-scale manner. , peptides, or antibiotics, amino acids, nucleic acids (such as DNA or RNA). Unless otherwise stated herein, the devices, equipment, and methods may include any desired capacity or production capacity, including, but not limited to, bench scale, pilot scale, and full production scale capacity. can.

Additionally, unless otherwise specified herein, the devices, equipment, and methods may include stirred tank, airlift, fiber, microfiber, hollow fiber, ceramic matrix, fluidized bed, fixed bed, and/or spouted bed bioreactors. Any suitable reactor can be included, including but not limited to. As used herein, a "reactor" can include a fermenter or fermentation unit, or any other reaction vessel, and the term "reactor" is used interchangeably with "fermenter." For example, in some embodiments, an exemplary bioreactor unit provides the following: supply of nutrients and/or carbon sources, injection of a suitable gas (e.g., oxygen), inflow and outflow of fermentation or cell culture media, one or more of separating gas and liquid phases, maintaining temperature, maintaining oxygen and _CO2 levels, maintaining pH levels, agitation (e.g., stirring), and/or cleaning/sterilization. Or you can do all of them. Exemplary reactor units, such as fermentation units, may include multiple reactors within the unit, e.g., the units may include 1, 2, 3, 4, 5, 10, 15, 20 reactors within each unit and/or facility. , 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, or 100 bioreactors, and/or have single or multiple reactors within the facility. It may include a plurality of units having. In various embodiments, the bioreactor may be suitable for batch, semi-fed-batch, fed-batch, perfusion, and/or continuous fermentation processes. Any suitable reactor diameter can be used. In embodiments, the bioreactor can have a volume of about 100 mL to about 50,000 L. Non-limiting examples include 100 mL, 250 mL, 500 mL, 750 mL, 1 liter, 2 liters, 3 liters, 4 liters, 5 liters, 6 liters, 7 liters, 8 liters, 9 liters, 10 liters, 15 liters, 20 liters. liter, 25 liter, 30 liter, 40 liter, 50 liter, 60 liter, 70 liter, 80 liter, 90 liter, 100 liter, 150 liter, 200 liter, 250 liter, 300 liter, 350 liter, 400 liter, 450 liter, 500 liters, 550 liters, 600 liters, 650 liters, 700 liters, 750 liters, 800 liters, 850 liters, 900 liters, 950 liters, 1000 liters, 1500 liters, 2000 liters, 2500 liters, 3000 liters, 3500 liters, 4000 liters , 4500 liters, 5000 liters, 6000 liters, 7000 liters, 8000 liters, 9000 liters, 10,000 liters, 15,000 liters, 20,000 liters, and/or 50,000 liters. Additionally, suitable reactors may be multi-use, single-use, disposable, or non-disposable, and may be made of stainless steel (e.g., 316L or any other suitable stainless steel) as well as Inconel, plastic, and/or glass. It may be formed from any suitable material including metal alloys such as.

In embodiments, and unless otherwise specified herein, the devices, equipment, and methods described herein also include any suitable unit operations and/or equipment not specifically mentioned, e.g. It may include operations and/or equipment for separation, purification, and separation. Any suitable equipment and environment may be used, such as conventional stick-built equipment, modular equipment, mobile equipment, and temporary equipment, or any other suitable construction, equipment, and/or layout. can. For example, in some embodiments a modular clean room may be used. Additionally, unless otherwise specified, the devices, systems, and methods described herein may be housed and/or performed in a single location or facility, or alternatively, in separate or multiple locations and/or facilities. or may be housed and/or carried out in a facility.

A suitable technique may include culturing in a bioreactor starting with a batch phase, followed by a short exponential fed-batch phase at a high specific growth rate, and further followed by a fed-batch phase at a low specific growth rate. Another suitable culture technique is a batch phase followed by a fed-batch phase at any suitable specific growth rate, or from a high growth rate to a low growth rate over the POI production time, or from a low growth rate to a high growth rate over the POI production time. Combinations of specific growth rates leading to growth rates can be included. Another suitable culture technique may include a batch phase followed by a continuous culture phase at low dilution rates.

Preferred embodiments include batch cultivation to provide biomass, followed by fed-batch cultivation for high yield POI production.

The host cells described herein are cultured in a bioreactor under growth conditions to provide at least 1 g/L cell dry weight, more preferably at least 10 g/L cell dry weight, preferably at least 20 g/L cell dry weight. It is preferred to obtain a cell density of cell dry weight, preferably at least 30, 40, 50, 60, 70, or 80 g/L dry weight. It would be advantageous to provide such yields of biomass production on a pilot or industrial scale.

Growth media (particularly basal growth media) that allow biomass accumulation typically include a carbon source, a nitrogen source, a sulfur source, and a phosphate source. Typically, such media further contain trace elements and vitamins, and may further contain amino acids, peptones, or yeast extract.

Preferred nitrogen sources include NH ₄ H ₂ PO ₄ , or NH ₃ , or (NH ₄ ) ₂ SO ₄ .
Preferred sulfur sources include MgSO ₄ , or (NH ₄ ) ₂ SO ₄ , or K ₂ SO ₄ .
Preferred phosphate sources include NH ₄ H ₂ PO ₄ , or H ₃ PO ₄ , or NaH ₂ PO ₄ , KH ₂ PO ₄ , Na ₂ HPO ₄ , or K ₂ HPO ₄ Further exemplary Media components include KCl, _CaCl2 , and the following trace elements: Fe, Co, Cu, Ni, Zn, Mo, Mn, I, B.
Preferably, the medium is supplemented with vitamins essential for growth, eg B vitamins such as _B7 .
P. A typical growth medium for P. pastoris contains glycerol, sorbitol or glucose, NH ₄ H ₂ PO ₄ , MgSO ₄ , KCl, CaCl ₂ , biotin, and trace elements.

In the production phase, the production medium, in particular, only a limited amount of supplementary carbon source is used.

Preferably, the host cell line is cultured in a mineral medium containing a suitable carbon source, which further simplifies the isolation process considerably. Examples of preferred mineral media include available carbon sources (e.g., glucose, glycerol, sorbitol, methanol, ethanol, or combinations thereof), macroelements (potassium, magnesium, calcium, ammonium, chlorides, sulfates, phosphates). salts) and trace elements (copper, iodide, manganese, molybdenum, cobalt, zinc, and iron salts, and boric acid), and optionally vitamins or amino acids (e.g. auxotrophic supplements). It is a medium containing.

Specifically, cells are cultured under conditions suitable to result in expression of the desired POI, and the POI is determined by the expression system and the nature of the expressed protein (e.g., whether the protein is fused to a signal peptide; Depending on whether the protein is soluble or membrane bound, it can be purified from cells or culture media. As will be appreciated by those skilled in the art, culture conditions will vary depending on factors including the host cell type and the particular expression vector used.

A typical production medium includes a supplemental carbon source, and also includes NH ₄ H ₂ PO ₄ , MgSO ₄ , KCl, CaCl ₂ , biotin, and trace elements.

For example, the supply of supplemental carbon source added to the fermentation may include a carbon source containing up to 50% available sugars, or up to 100% available alcohol by weight.

Fermentation is preferably carried out at a pH in the range 3-8.

Typical fermentation times are about 24-120 hours at temperatures ranging from 20°C to 35°C, preferably 22-30°C.

The POI is expressed using conditions to produce a yield of at least 1 mg/L, preferably at least 10 mg/L, preferably at least 100 mg/L, most preferably at least 1 g/L.

The term "expression" or "expression cassette" refers to containing the desired coding sequences (referred to herein as genes) and controlling sequences in operable linkage (so that transformation with these molecules is possible). or a transfected host is understood to refer to a nucleic acid molecule (also referred to herein as a polynucleotide) capable of incorporating the respective sequence and producing the encoded protein or host cell metabolite. . The term "expression" as used herein refers to the expression of a polynucleotide or gene, or the respective polypeptide or protein.

One or more expression cassettes are also understood herein as an "expression system." The expression system may be included in an expression construct such as a vector, but the relevant DNA may be integrated into the chromosome of the host cell. Expression can refer to secreted or non-secreted expression products, including polypeptides or metabolites.

Expression cassettes are conveniently provided as expression constructs, e.g. in the form of "vectors" or "plasmids," which typically contain cloned recombinant nucleotide sequences (i.e., recombinant DNA sequences required for transcription of genes (genes) and translation of their mRNA. The expression vector or plasmid usually contains an origin for autonomous replication in the host cell, or a genetic locus for genomic integration, a selectable marker (e.g., an amino acid synthesis gene, or zeocin, kanamycin, G418, or hygromycin). , a gene conferring resistance to antibiotics such as noseothricin), several restriction enzyme cleavage sites, a suitable promoter sequence, and a transcription terminator, these components being operably linked together. As used herein, the terms "plasmid" and "vector" include genomes that incorporate autonomously replicating nucleotide sequences, as well as artificial chromosomes, such as yeast artificial chromosomes (YACs).

Expression vectors can include, but are not limited to, cloning vectors, modified cloning vectors, and specially designed plasmids. Preferred expression vectors described herein are expression vectors suitable for the expression of recombinant genes in eukaryotic host cells, and are selected depending on the host organism. A suitable expression vector typically contains suitable regulatory sequences for expressing the POI-encoding DNA in a eukaryotic host cell. Examples of regulatory sequences include promoters, operators, enhancers, ribosome binding sites, and sequences that control initiation and termination of transcription and translation. Regulatory sequences are typically operably linked to the DNA sequence to be expressed.

To enable expression of a recombinant nucleotide sequence in a host cell, a promoter sequence typically regulates and initiates transcription of downstream nucleotide sequences to which it is operably linked. The expression cassette or vector typically includes promoter nucleotide sequences upstream and adjacent to the coding sequence (e.g., encoding a helper factor) or gene of interest (GOI), adjacent to the 5' end of the coding sequence. or, if signal or leader sequences are used, upstream of the signal and leader sequences, respectively, to facilitate translation initiation and expression of the coding sequence to yield the expression product (e.g., TIF or POI). contains adjacent promoter nucleotide sequences.

Certain expression constructs described herein include a promoter operably linked to a nucleotide sequence encoding TIF or POI under the transcriptional control of the promoter. Specifically, promoters can be used that are not naturally associated with the coding sequence in question.

The specific expression constructs described herein include a leader sequence (e.g., secretory signal peptide sequence (pre-sequence), or pro-sequence) that causes transport of the POI into the secretory pathway and/or secretion of the POI from the host cell. a polynucleotide encoding a POI linked to a POI. The presence of such a secretory leader sequence in the expression vector is typically required when the POI intended for recombinant expression is a protein that is not naturally secreted and thus lacks a natural secretory leader sequence. or if the nucleotide sequence has been cloned without its natural secretory leader sequence. Generally, any secretion leader sequence effective to cause secretion of the POI from the host cell can be used. The secretory leader sequence may be derived from yeast sources, such as yeast alpha factor (eg, MFα of Saccharomyces cerevisiae) or yeast phosphatase, from mammalian or plant sources, or otherwise.

In certain embodiments, multiple cloning vectors can be used, which are vectors that have multiple cloning sites. Specifically, a desired heterologous polynucleotide can be incorporated or incorporated into a multiple cloning site to prepare an expression vector. For multiple cloning vectors, the promoter is typically placed upstream of the multiple cloning site.

As used herein, the term "gene expression" or "expressing a polynucleotide" or "expressing a nucleic acid molecule" refers to the transcription of DNA into mRNA, translation and processing of mRNA, maturation of mRNA, It is meant to include at least one step selected from the group consisting of mRNA transport, protein folding and/or protein transport.

The terms "polynucleotide," "nucleic acid molecule," or "nucleic acid sequence" are used interchangeably herein and refer to either ribonucleotides or deoxyribonucleotides, in polymeric unbranched form of any length, or Refers to both combinations of nucleotides. Preferably, polynucleotide refers to deoxyribonucleotides of any length in polymeric unbranched form. Here, a nucleotide consists of a pentose sugar (deoxyribose), a nitrogenous base (adenine, guanine, cytosine, or thymine), and a phosphate group.

As used herein, the term "co-express" or "co-expression" refers to the expression of at least two or more polynucleotides (nucleic acid molecules, such as genes) in a host cell, cell line, or cell culture. For example, concomitant or sequential (while still culturing cells in the same cell culture or enclosure) or simultaneous expression in approximately the same or different amounts or proportions.

As described herein, polynucleotides (such as a TIF gene) can be co-expressed such that at least one of the polynucleotides (such as a GOI) is overexpressed.

Host cells that co-express a TIF gene as described herein are specifically genetically engineered and modified to increase expression of the TIF gene in host cell culture. This is also referred to herein as "overexpression" or "co-overexpression."

As used herein, the term "overexpress" or "overexpression" refers to expression of the same expression product at a higher level or under defined conditions than the expression of the same expression product prior to the genetic modification of the host cell. refers to the expression of an expression product (e.g., a polypeptide or protein) in a non-equivalent host. It is understood that a TIF that is heterologous to a host cell is overexpressed whenever such a host cell expresses such TIF. For example, if the host cells described herein do not naturally express any of the TIFs of interest, a heterologous polynucleotide encoding such TIF protein is de novo introduced into the host cell for expression; Detectable expression of TIF is encompassed by the term "overexpression."

Overexpression of a gene encoding a protein (eg, TIF gene) is also referred to as overexpression of the protein (eg, TIF). Overexpression can be achieved in any manner known to those skilled in the art. Generally, it can be achieved by increasing the transcription/translation of the gene, for example by increasing the copy number of the gene, or by changing or modifying regulatory sequences or sites associated with the expression of the gene. For example, a gene can be operably linked to a strong promoter to reach high expression levels. Such a promoter may be an endogenous promoter or a heterologous promoter (especially a recombinant promoter). The promoter can be replaced with a heterologous promoter that increases expression of the gene. The use of inducible promoters further allows for increased expression during the culture process. Additionally, overexpression can be achieved by, for example, altering the chromosomal location of a particular gene, changing the nucleic acid sequence (e.g., ribosome binding site or transcription terminator) flanking a particular gene, or introducing a frameshift in the open reading frame. modifying proteins involved in transcription of genes and/or translation of gene products (e.g., regulatory proteins, suppressors, enhancers, transcription activators, etc.), or altering the expression of certain gene routines in the art. Any other conventional means of modulating (e.g., blocking the expression of a repressor protein or deleting or mutating the gene for a transcription factor that normally suppresses the expression of the gene desired to be overexpressed, antisense) (including, but not limited to, the use of nucleic acid molecules). Expression levels can also be improved by extending the lifespan of the mRNA. For example, specific terminator regions can be used to extend the half-life of mRNA. If multiple copies of the gene are included, the gene can be placed on plasmids with different copy numbers or integrated into the chromosome and amplified. One or more genes or genomic sequences can be introduced into a host cell for expression.

According to certain embodiments, each TIF-encoding polynucleotide may be presented in a single copy or in multiple copies per cell. The copies may be adjacent or separate from each other. According to another specific embodiment, overexpression of each TIF uses a recombinant nucleotide sequence encoding a TIF, wherein the TIF is one suitable for integration (i.e., knock-in) into the genome of the host cell. Provided in single or multiple copies per cell on the above plasmids. The copies may be adjacent or separate from each other. Overexpression can be achieved by expressing multiple copies of the polynucleotide, eg, 2, 3, 4, 5, 6 or more copies of the polynucleotide per host cell.

Recombinant nucleotide sequences comprising polynucleotides (genes) encoding GOIs and their respective TIFs may be provided on one or more autonomously replicating plasmids and may be introduced in single or multiple copies per cell. It's okay.

Alternatively, the recombinant nucleotide sequences comprising polynucleotides (genes) encoding GOI and TIF may be present on the same plasmid and may be introduced in single or multiple copies per cell.

The heterologous polynucleotide (gene) encoding the respective TIF, or the heterologous recombinant expression construct containing the polynucleotide (gene) encoding the TIF, is preferably integrated into the genome of the host cell.

The term "genome" generally refers to the entire genetic information of an organism encoded in DNA (or RNA). It may be present on the chromosome, on a plasmid or vector, or both. Preferably, the polynucleotide (gene) encoding each TIF is integrated into the chromosome of the cell.

The polynucleotide (gene) encoding each TIF can be integrated into its natural locus. "Natural locus" refers to the specific chromosomal location at which the polynucleotide (gene) encoding TIF is located in naturally occurring wild-type cells. However, in another embodiment, the polynucleotide (gene) encoding TIF is present in the host cell's genome where it has been ectopically integrated, rather than at its natural locus. The term "ectopic integration" refers to the insertion of a nucleic acid into the genome of a microorganism at a site other than its normal chromosomal locus (ie, scheduled or random integration). In another embodiment, the polynucleotide (gene) encoding TIF is integrated into the natural locus and ectopically. Heterologous recombination can be used to achieve random or untargeted integration. Heterologous recombination refers to recombination between DNA molecules with significantly different sequences.

In certain embodiments, polynucleotides (genes) encoding each TIF and/or GOI can be incorporated into a plasmid or vector. Preferably the plasmid is a eukaryotic expression vector, preferably a yeast expression vector. Suitable plasmids or vectors are described further herein.

Overexpression of endogenous or heterologous polynucleotides in recombinant host cells can be achieved by modifying expression control sequences. Expression control sequences are known in the art and include, for example, promoters, enhancers, polyadenylation signals, transcription terminators, internal ribosome entry sites (IRES), etc. that provide for expression of the polynucleotide sequence in a host cell. Expression control sequences interact specifically with cellular proteins involved in transcription. Exemplary expression control sequences are described, for example, in Goeddel, Gene Expression Technology: Methods in Enzymology, Vol. 185, Academic Press, San Diego, Calif. (1990).

In a preferred embodiment, overexpression is achieved by expressing the polynucleotide using an enhancer. Transcriptional enhancers are found 5' and 3' of transcription units, within introns as well as within the coding sequence itself, and are relatively orientation and position independent. Enhancers can be spliced into the expression vector at positions 5' or 3' of the coding sequence, but are preferably located 5' from the promoter. Most yeast genes contain only one UAS, typically located within a few hundred base pairs of the CAP site, and most yeast enhancers (UASs) are nonfunctional when located 3′ of the promoter, but , higher eukaryotic enhancers can function both 5' and 3' of a promoter.

A large number of enhancer sequences from mammalian genes (globin, RSV, SV40, EMC, elastase, albumin, a-fetoprotein, and insulin) are known. Additionally, enhancers derived from eukaryotic viruses such as the SV40 late enhancer, the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and the adenovirus enhancer may be used.

Specifically, the GOI and/or TIF-encoding polynucleotides (genes) described herein are operably linked to transcriptional and translational regulatory sequences that provide for expression in the host cell. As used herein, the term "translation control sequence" refers to a nucleotide sequence that is associated with a gene nucleic acid sequence and regulates translation of the gene. Transcriptional and/or translational control sequences can either be placed on a plasmid or vector, or integrated into the chromosome of the host cell. A transcriptional and/or translational control sequence is located on the same nucleic acid molecule of the gene it regulates.

Specifically, overexpression of each TIF was performed as described by Marx et al. , 2008 (Marx, H., Mattanovich, D. and Sauer, M. Microb Cell Fact 7 (2008): 23) by methods known in the art (e.g., endogenous regulatory regions). (by genetically modifying the gene); such methods include, for example, the incorporation of recombinant promoters to increase expression of the gene.

For example, overexpression of an endogenous or heterologous polynucleotide in a recombinant host cell can be achieved by modifying the promoter that controls such expression, e.g., to reach high expression levels, the polynucleotide is operably linked to the polynucleotide. This can be accomplished by replacing a strong promoter (eg, the endogenous promoter or promoter naturally linked to the polynucleotide in a wild-type organism) with another stronger promoter. Such promoters may be inducible or constitutive. Promoter modifications can also be performed by mutagenesis methods known in the art.

A specific embodiment refers to co-expression of TIF (or TIF gene) in conjunction with expressing GOI. In some embodiments described herein, a vector or nucleic acid sequence can include one or more expression cassettes for co-expressing at least one TIF molecule and a GOI. Vectors or nucleic acid sequences can be combined into two or more vectors using a number of techniques, including co-transfection of two or more plasmids, the use of multiple promoters or bidirectional promoters, or the creation of bicistronic or multicistronic vectors. can be constructed to allow co-expression of polynucleotides of

Specific embodiments, for example, stably integrate at least 1, 2, 3, 4, or 5 TIFs in introducing their respective expression cassettes for stable integration within the genome or chromosome of a host cell. Refers to genetic modification for co-expression.

As used herein, the term "functional variant" is also referred to as "functionally active variant", e.g., a naturally occurring variant derived from or related to TIF, a nucleotide sequence or an amino acid sequence of TIF. Any sequence other than the sequence ("native" being understood as the sequence that naturally occurs in a wild-type cell) is meant. Specific functional variants of any of the (parental) TIF or respective TIF genes with specific sequence identity to the parent sequence (e.g., any one of SEQ ID NOs: 1 to 65, in particular the sequences 1, 12, 23, 34, 45, or 56).

According to certain embodiments, a functional variant is derived from a native sequence and comprises or consists of a given sequence that has a demonstrated function in a host cell, and which, compared to the native sequence from which it is derived, has approximately Same and/or improved.

According to certain embodiments, a functional variant is an isoform or ortholog of a naturally occurring parent molecule, and an ortholog is a species other than a species (e.g., a mammalian species or a fungal species) that contains the naturally occurring parent molecule. Naturally occurring in the species.

In some embodiments, functional variants of polynucleotides or nucleic acid molecules improve, e.g., improve nucleic acid stability, increase translation efficiency in host cells, reduce the number of truncated proteins expressed, The sequence has been optimized to improve folding or prevent misfolding, reduce toxicity of the expressed product, reduce cell death caused by the expressed product, or increase and/or reduce protein aggregation. Contains nucleotide sequences. According to certain embodiments, functional variants of a parent nucleotide sequence are expressed in a host cell, obtainable by one or more genetic modifications of the parent nucleotide sequence for improved expression in the cellular environment of the host cell. is a codon-optimized variant of the parent nucleotide sequence in question.

The functional variants of TIF described herein have substantially the same or improved activity as the native sequence (particularly to improve production of POIs when co-expressed in host cells) , is considered functionally active.

A functionally active variant of TIF is produced by producing the variant by mutagenesis of the respective native (wild type) TIF gene, expressing the variant concomitantly or simultaneously with a gene encoding a heterologous POI in a host cell, and producing a variant. POI can be prepared by evaluating the activity of POI and improving the productivity of host cells to produce POI.

The activity of TIF can be determined as described by well-known methods, eg, using in vitro and in vivo approaches.

A suitable method for analyzing translation activity is described by Dermit et al. (Mol Biosyst. 2017 Nov 21; 13(12): 2477-24882017).

Some additional suitable methods are described by Martin R (1998; Protein synthesis: methods and protocols. Methods in Molecular Biology, Volume 77, Totowa, N.J.: Humana actively, as described by Press) Radiolabeling of the protein to be translated (incorporation of radiolabeled amino acids) is used.

Specific tests to measure translation activity are described in the Examples section below.

Functional variants of a parent protein include, for example, proteins in which one or more amino acid residues are added or deleted at the N-terminus or C-terminus, as well as within one or more internal domains. Certain functionally active variants subdivide the parent sequence by, for example, less than 100 amino acids, specifically less than 75 amino acids, more specifically less than 50 amino acids, more specifically less than 25 amino acids, or otherwise Include additional amino acids at the N-terminus and/or C-terminus to extend by less than 10 amino acids. Further particular functionally active variants may be fusion proteins, in which the sequences of the invention are extended by additional amino acid residues of another polypeptide or protein.

A particular functional variant is a fragment of a parent protein or nucleic acid molecule.

A functional variant that is a fragment of a polynucleotide or nucleic acid molecule can range from at least 20 nucleotides, preferably at least 100 nucleotides, to the full-length nucleotide sequence encoding the TIF described herein. A functionally active fragment of a polynucleotide or nucleic acid molecule may comprise at least 50%, preferably at least any of 60, 70, 80, 85, 90%, or 95% of the respective nucleotide sequence.

A functional variant that is a fragment of a polypeptide or protein has at least 10 amino acids, specifically at least 25 amino acids, more specifically at least 50 amino acids, more specifically at least 75 amino acids. , or at least 100 contiguous amino acids, or up to the total number of amino acids present in the full-length protein.

As used herein, the term "endogenous" refers to a wild type (natural) host prior to modification to reduce expression of the respective endogenous gene and/or to reduce production of an endogenous protein. It is meant to include those molecules and sequences (especially endogenous genes or proteins) that are present in the cell. In particular, an endogenous nucleic acid molecule (e.g., a gene) or protein that is present in (and can be obtained from) a particular host cell is "endogenous to the host cell" or "intrinsic to the host cell" because it is found in nature. It is understood to be ``sexuality.'' Furthermore, a cell that "endogenously expresses" a nucleic acid or protein expresses that nucleic acid or protein as in the same particular type of host in which it is found in nature. Additionally, a host cell is "endogenously producing" a nucleic acid, protein, or other compound, or a host cell "endogenously producing" a host cell of the same particular type as found in nature. Like a cell, it produces its nucleic acid, protein, or compound.

Thus, even if an endogenous protein is no longer produced by a host cell, such as a knockout mutant of the host cell in which the gene encoding the protein has been inactivated or deleted, the protein is still referred to herein as "endogenous." It is called.

As used herein with respect to a nucleotide sequence, construct (e.g., expression cassette), amino acid sequence, or protein, the term "heterologous" refers to a nucleotide sequence, construct (e.g., expression cassette), amino acid sequence, or protein that is foreign to a given host cell (i.e., be "exogenous", such that the host cell is not found (e.g., "endogenous"), or found naturally in a given host cell (e.g., "endogenous"), but in the context of a heterologous construct or incorporated into such a heterologous construct. Refers to a compound that is either fused with an endogenous nucleic acid or used in conjunction with an endogenous nucleic acid to render the construct heterologous. A heterologous nucleotide sequence found endogenously may also be produced in an unnatural (eg, greater than expected or greater than that found in nature) amount in a cell. A heterologous nucleotide sequence, or a nucleic acid comprising a heterologous nucleotide sequence, likely differs in sequence from the endogenous nucleotide sequence, but encodes the same protein as found endogenously. Specifically, a heterologous nucleotide sequence is one that is not found in the same relationship with a host cell in nature. It is understood that any recombinant or artificial nucleotide sequence is heterologous. Examples of heterologous polynucleotides include nucleotide sequences that are not naturally associated with a promoter (e.g., to obtain a hybrid promoter) or are not operably linked to a coding sequence, as described herein. A nucleotide sequence. As a result, a hybrid polynucleotide or a chimeric polynucleotide can be obtained. A further example of a heterologous compound is a polynucleotide encoding a POI that is operably linked to a transcriptional control element (e.g., a promoter) to which the endogenous naturally occurring POI-encoding sequence is not normally operably linked. It is a nucleotide.

The term "translation initiation factor", abbreviated "TIF", as used herein refers to a translation initiation factor protein or a polynucleotide (nucleic acid molecule) encoding a translation initiation factor.

Specifically, none of the TIFs described herein are proteins of interest (POI). The recombinant host cells described herein are expression systems that express a TIF and further express another polynucleotide (different from the TIF encoding a polynucleotide), referred to as a gene of interest (GOI). is particularly understood to include.

As used herein, the term "translation initiation factor" specifically refers to any factor included in an mRNP.

The nucleotide sequence encoding a specific TIF can be derived from the naturally occurring sequence or a functionally active variant thereof (e.g. by nucleotide sequence optimization algorithms or by codon optimization to improve its expression in recombinant host cells). optimizing the sequence by techniques (a variant nucleotide sequence that differs from the parent (naturally occurring) by one or more point mutations, e.g., up to any of 50, 40, 30, 20, or 10 point mutations); .

Particular optimization techniques are to improve the expression of a nucleotide sequence in a host cell, for example by nucleotide sequence optimization algorithms or by codon optimization techniques.

Certain optimization techniques have improved cloning, such as Golden Gate cloning and Golden Gate assembly optimization.

Particular optimized nucleotide sequences include, for example, "silent" mutations to avoid the presence of recognition sites for any restriction enzymes used (eg, BsaI and Bpil).

The optimized nucleotide sequences described herein typically include one or more, e.g., several point mutations in the encoded amino acid sequence, e.g., up to 10, 9, 8, 7, 6, Allows for 5, 4, or 3 point mutations.

"eIF4E", also known as eukaryotic translation initiation factor 4E, is a TIF involved in the formation of closed-loop mRNA; it is a closed-loop factor, part of the eIF4F cap-binding complex, and part of the mRNA It is. It is characterized by any one of SEQ ID NOs: 1-11 or orthologs in other eukaryotic species. eIF4E is encoded by a nucleotide sequence encoding eIF4E, which may be a naturally occurring eIF4E gene, or a respective functional variant thereof encoding eIF4E with specific sequence identity to naturally occurring eIF4E. An exemplary eIF4E-encoding nucleotide sequence is specified by SEQ ID NO: 66 of Komagataella phaffii, which encodes SEQ ID NO: 1, or a functionally active variant thereof. SEQ ID NO: 67 identifies an example of an optimized coding nucleotide sequence that has been produced by Golden gate optimization and differs from SEQ ID NO: 66 by three nucleotide substitutions: A276G, T354C, G492A.

eIF4A, also known as eukaryotic translation initiation factor 4A, is a TIF involved in the formation of closed-loop mRNA; it is a closed-loop factor, part of the eIF4F cap-binding complex, and part of the mRNA . It is characterized by any one of SEQ ID NOs: 12-33 or orthologs in other eukaryotic species. The term "eIF4A" includes TIF2a and TiF2b, with TIF2b being a longer variant of TIF2a of 249 nucleotides (corresponding to 83 amino acids) on the 5' end. Both sequences are P. presented two variants of eIF4A that were directly amplified from the P. pastoris genome and have alternative starting positions.

eIF4A is encoded by a nucleotide sequence encoding eIF4A, which may be a naturally occurring eIF4A gene, or a respective functional variant thereof encoding eIF4A with specific sequence identity to naturally occurring eIF4A. Exemplary eIF4A-encoding nucleotide sequences are specified by SEQ ID NO: 68 or SEQ ID NO: 70 of Komagataella phaffii encoding SEQ ID NO: 12 and SEQ ID NO: 23, respectively, or are functionally active variants thereof. SEQ ID NO: 69 identifies an example of an optimized coding nucleotide sequence that has been produced by Golden gate optimization and differs from SEQ ID NO: 68 by one nucleotide substitution: C45A. SEQ ID NO: 71 identifies an example of an optimized coding nucleotide sequence that has been produced by Golden gate optimization and differs from SEQ ID NO: 70 by one nucleotide substitution: C294A.

eIF4G, also known as eukaryotic translation initiation factor 4G, is a closed-loop factor, a TIF that is part of the eIF4F cap-binding complex and involved in the formation of closed-loop mRNA, which is part of the mRNP. It is characterized by any one of SEQ ID NOs: 34-44 or its orthologues in other eukaryotic species. eIF4G is encoded by a nucleotide sequence encoding eIF4G, which may be a naturally occurring eIF4G gene, or a respective functional variant thereof encoding eIF4G with specific sequence identity to naturally occurring eIF4G. An exemplary eIF4G-encoding nucleotide sequence is specified by SEQ ID NO: 72 of Komagataella phaffii, which encodes SEQ ID NO: 34, or a functionally active variant thereof. SEQ ID NO: 73 identifies an example of an optimized coding nucleotide sequence that has been produced by Golden gate optimization and differs from SEQ ID NO: 72 by four nucleotide substitutions: A564G, A1923G, G2037C, T2100C.

PAB1, also known as polyadenylate binding protein 1, is a TIF that is involved in the formation of closed-loop mRNA and part of mRNA. It is characterized by any one of SEQ ID NOs: 45-55, or orthologs in other eukaryotic species. PAB1 is encoded by a nucleotide sequence encoding PAB1, which may be the naturally occurring PAB1 gene, or its respective functional variant encoding PAB1 with specific sequence identity to naturally occurring PAB1. An exemplary PAB1-encoding nucleotide sequence is specified by SEQ ID NO: 74 of Komagataella phaffii, which encodes SEQ ID NO: 45, or a functionally active variant thereof. SEQ ID NO: 75 identifies an example of an optimized coding nucleotide sequence that has been produced by Golden gate optimization and differs from SEQ ID NO: 74 by three nucleotide substitutions: C150A, T384C, C707T.

RLI1, also known as ATP-binding cassette subfamily E member 1 (ABCE1) and RNaseL inhibitor (RLI), is an ATP-binding cassette (ABC) protein encoded by the ABCE1 gene in humans. This is a TIF that has dual roles in translation initiation and ribosome biogenesis as well as translation termination and part of the mRNP. It is characterized by any one of SEQ ID NOs: 56-65 or orthologs in other eukaryotic species. RLI1 is encoded by a nucleotide sequence encoding RLI1, which may be a naturally occurring RLI1 gene, or a respective functional variant thereof encoding RLI1 with specific sequence identity to naturally occurring RLI1. An exemplary RLI1-encoding nucleotide sequence is specified by SEQ ID NO: 76 of Komagataella phaffii, which encodes SEQ ID NO: 56, or a functionally active variant thereof.

Comprising the amino acid sequences specified by SEQ ID NOs: 1, 12, 23, 34, 45, and 56 (including the (optimized) nucleotide sequences of each) provided herein and used in the Examples section or a TIF consisting of K. phaffii origin. TIFs comprising or consisting of the amino acid sequences specified by SEQ ID NOs: 2, 13, 24, 35, 46, and 57 provided herein are those of K. pastoris origin. It is well understood that homologous sequences exist in other yeast host cells (particularly methylotrophic yeast), such as those provided in Figure 1, that can be used as described herein. There is. For example, the yeast Pichia pastoris contains respective homologous sequences. Pichia pastoris has been reclassified into the genus Komagataera, and K. pastoris, K. phaffii, and K. pseudopastoris is divided into three species.

For example, any homologous sequence of each TIF with the specific sequence identity described herein, particularly any homologous sequence of each P. Any such protein that is an ortholog of TIF of K. pastoris (eg, K. phaffii, K. pastoris, or K. pseudopastoris) can be used.

The term "mRNP" as used herein refers to messenger RNP (messenger ribonucleoprotein), understood as a particle or complex consisting of mRNA with binding proteins. mRNA is bound by a variety of proteins during synthesis, splicing, transport, and translation in the cytoplasm.

As used herein, the term "operably linked" means such that the function of one or more nucleotide sequences is affected by at least one other nucleotide sequence present in the nucleic acid molecule. , refers to the association of nucleotide sequences in a single nucleic acid molecule (eg, a vector or expression cassette). A nucleic acid sequence is placed into a functional relationship with another nucleic acid sequence on the same nucleic acid molecule by operably linking. For example, a promoter is operably linked to a coding sequence of a recombinant gene if it is capable of affecting expression of the coding sequence. As a further example, a nucleic acid encoding a signal peptide is operably linked to a nucleic acid sequence encoding a POI if it is capable of expressing a secreted form of the protein, such as the mature protein or a pre-form of the mature protein. In particular, such nucleic acids operably linked to each other can be directly linked, ie, without additional elements or nucleic acid sequences between the signal peptide-encoding nucleic acid and the POI-encoding nucleic acid sequence. Alternatively, suitable linking sequences may be used, such as, for example, a cloning site located between the promoter and the GOI.

A "promoter" sequence is typically understood to be operably linked to a coding sequence when the promoter controls transcription of the coding sequence. If a promoter sequence is not naturally associated with a coding sequence, its transcription is either not controlled by the promoter in the native (wild type) cell or the sequence has recombined with a different contiguous sequence. It is.

Promoters are described herein to initiate, regulate, or otherwise mediate or control the expression of a protein encoding a polynucleotide (DNA), such as a DNA encoding a POI. Promoter DNA and code DNA may be derived from the same gene or different genes, and may be derived from the same or different organisms.

Promoter strength specifically refers to its transcriptional strength as expressed by the efficiency of initiation of transcription that occurs at that promoter at high or low frequency. The higher the transcription strength, the more frequently transcription will occur at that promoter. Promoter strength is a typical characteristic of promoters. This is because promoters determine how often a given mRNA sequence is transcribed, effectively giving higher priority to the transcription of some genes than others, resulting in higher concentrations of transcripts. It is from. For example, genes encoding proteins that are required in large quantities typically require relatively strong promoters. RNA polymerase can only perform one transcription task at a time, so it must prioritize to be efficient. To enable this prioritization, differences in promoter strength are selected.

Promoter strength can also refer to the frequency of transcription, commonly understood as the rate of transcription, as determined, for example, by the amount of transcript in a suitable assay (eg, RT-PCR or Northern blotting). For example, the transcriptional strength of the promoters described herein is determined by the transcriptional strength of the promoters described herein. P. pastoris was determined in host cells. compared to the native pGAP promoter of P. pastoris.

The strength of a promoter to express a gene of interest is generally understood as the strength of expression or the ability to support high expression levels/rates. For example, the expression and/or transcription strength of the promoter of the invention may be determined by P. P. pastoris was determined in host cells. pastoris native pGAP promoter, and measured, for example, when fully induced or derepressed.

According to certain embodiments, the GOIEC promoter is stronger than the TIFEC promoter. Preferably, the rate or strength of transcription, as determined in the host cell (e.g. eukaryote) chosen as the host cell for recombinant purposes to produce the POI, as compared to the native pGAP promoter, is At least one of 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%, or even more, and 110%, 120%, 130 %, 140%, 150%, 160%, 170%, 180%, 190%, or 200%, or even more. The expression rate can be determined, for example, by the expression level of a reporter gene such as eGFP.

The natural pGAP promoter typically initiates expression of the GAP gene encoding glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a constitutive promoter present in most organisms. GAPDH (EC 1.2.1.12) is a key enzyme in sugar enzymatic degradation and gluconeogenesis, and plays an important role in catabolic and anabolic carbohydrate metabolism.

The relative transcriptional strength compared to a reference promoter can be determined by standard methods, e.g., by measuring the amount of transcript using a microarray, or by measuring the amount of the respective gene expression product in recombinant cells. It can be determined in cell culture by e.g. In particular, transcription rate can be determined by transcription intensity in microarrays, Northern blots, or quantitative real-time PCR (qRT-PCR), or RNA sequencing (RNA-seq).

As described herein, the heterologous promoter is used to express any one or more or all of the TIFs in each TIFEC and/or to express the GOI in the GOIEC. be able to. A heterologous promoter is a polynucleotide to be expressed and/or an artificial promoter, or a promoter derived from a wild-type host cell, but placed in the host cell genome within a heterologous expression cassette or at a location that does not naturally occur in the wild-type host cell. It may be heterologous to the promoter in which it is placed.

As described herein, according to certain embodiments, either the TIF expression cassette or the GOIEC comprises a constitutive promoter, e.g., any of the promoters further described herein. , can be used.

Specific examples of constitutive promoters include, for example, pGAP (e.g., SEQ ID NO: 100, SEQ ID NO: 101) and its functional variants, pCS1 (e.g., SEQ ID NO: 102, or its functional variants as disclosed in WO2014/139608). variant), pMDH3 (e.g., SEQ ID NO: 103), pPOR1 (e.g., SEQ ID NO: 104), pRPP1B, pPDC1, pGPM1, pFBA1-1, or a functional variant of any of the foregoing. .

Specific examples of inducible or repressible promoters include, for example, natural pAOX1 or pAOX2 and functional variants thereof, any of the regulatable promoters such as pG1-pG8 or fragments thereof published in WO2013/050551, and WO2017/ 021541A1, such as pG1 and pG1-x.

In particular, regulatable promoters such as derepressible or repressible (referred to herein as (de)repressible) or inducible promoters, such as the natural methanol-inducible promoters pAOX1 (SEQ ID NO: 81) or pAOX2 ( SEQ ID NO: 82), or P. any of the natural methanol-inducible promoters of P. pastoris (e.g., SEQ ID NOs: 83-96, published by Gasser, Steiger, & Mattanovich, Microb Cell Fact. 2015, 14:196), or any other carbon source. Regulatable promoters (e.g., derepressible promoters such as pG1-pG8 (pG1: SEQ ID NO: 97, pG3: SEQ ID NO: 105, pG4: SEQ ID NO: 106, pG5: SEQ ID NO: 107, pG7: SEQ ID NO: 108, pG8: SEQ ID NO: 109) promoter) and functional variants (e.g. fragments) of any of the foregoing (e.g. fragments of pG1 designated pG1a to pG1f: SEQ ID NOS: 110-115), as well as those published in WO2013/050551 and WO2017/021541 Functional variants designated pG1-x, in particular pG1-3 (eg, SEQ ID NO: 98, designated pG1-D1240) or pG1-4 (eg, SEQ ID NO: 99, designated pG1-D1427) or any of the foregoing functional variants having a length of at least 300, 400, or 500 bp (particularly including the 3' end), or any of the foregoing functional variants may be used.

In particular, a functional variant of a promoter described herein may be a functional variant of the promoter from which it is derived over the entire length or portion of the 3' end of the promoter sequence (where the portion is at least 300, 400, or 500 bp in length). have at least one of 80%, 85%, 90%, 95%, or 100% sequence identity, particularly with approximately the same promoter activity (e.g. , +/-50%, 40%, 30%, 20%, or 10%), but the promoter activity is relative to the promoter from which it is derived. can be improved. As shown in Figure 1, specific functional promoter variants of pG1-3 or pG1-4 include at least two major regulatory regions, and/or at least two core regulatory regions, and/or at least two T motifs. This includes:

Further examples of suitable promoter sequences are described in Prielhofer et al. (BMC Syst Biol. 2017.11(1):123) and Mattanovich et al. Triose phosphate isomerase (TPI), phosphoglycerate kinase (PGK), glyceraldehyde-3-phosphate dehydrogenase (GAPDH or GAP) and its variants, lactase (LAC) and galactosidase (GAL), P. P. pastoris glucose-6-phosphate isomerase promoter (PPGI), 3-phosphoglycerate kinase promoter (pPGK), glycerolaldehyde phosphate dehydrogenase promoter (pGAP), translation elongation factor promoter (PTEF), and P. pastoris. P. pastoris enolase 1 (pEN01), triose phosphate isomerase (pTPI), ribosomal subunit proteins (pRPS2, pRPS7, pRPS31, pRPL1), alcohol oxidase promoter (pAOX1, pAOX2) or its variant with modified properties, formaldehyde dehydrogenase promoter (pFLD) ), isocitrate lyase promoter (pICL), α-ketoisocaproate decarboxylase promoter (pTHI), promoters of heat shock protein family members (pSSA1, pHSP90, pKAR2), 6-phosphogluconate dehydrogenase (pGND1), phosphoglycerate mutase (pGPM1), transketolase (pTKL1), phosphatidylinositol synthase (pPIS1), ferro-02-oxidoreductase (pFET3), high-affinity iron permease (pFTR1), inhibitory alkaline phosphatase (pPH08), N-myristoyltransferase ( pNMT1), pheromone-responsive transcription factor (pMCM1), ubiquitin (pUBI4), single-stranded DNA endonuclease (pRAD2), promoter of the main ADP/ATP carrier of the mitochondrial inner membrane (pPET9) (WO2008/128701), and formate dehydrogenation Enzyme (FMD) promoter is mentioned.

Further examples of suitable promoters include S. S. cerevisiae enolase (ENO1), S. cerevisiae enolase (ENO1). S. cerevisiae galactokinase (GAL1), S. cerevisiae galactokinase (GAL1); S. cerevisiae alcohol dehydrogenase, and S. cerevisiae alcohol dehydrogenase. S. cerevisiae glyceraldehyde-3-phosphate dehydrogenase (ADH1, ADH2, GAP), S. cerevisiae glyceraldehyde-3-phosphate dehydrogenase (ADH1, ADH2, GAP). S. cerevisiae triose phosphate isomerase (TPI). S. cerevisiae metallothionein (CUP1), and S. cerevisiae metallothionein (CUP1). cerevisiae3-phosphoglycerate kinase (PGK), as well as the maltase gene promoter (MAL).

The terms "nucleotide sequence" or "nucleic acid sequence" as used herein refer to either DNA or RNA. A "nucleic acid sequence" or "polynucleotide sequence" or simply "polynucleotide" refers to a single- or double-stranded polymer of deoxyribonucleotides or ribonucleotide bases read from the 5' end to the 3' end. This includes expression cassettes, self-replicating plasmids, infectious polymers of DNA or RNA, as well as non-functional DNA or RNA.

The term "protein of interest" (POI) as used herein refers to a polypeptide or protein produced by recombinant techniques in a host cell. More specifically, the protein may be a polypeptide that is not naturally occurring in the host cell (i.e., a heterologous protein) or may be otherwise native to the host cell (i.e., a protein homologous to the host cell). ), for example, by transformation or transfection with a self-replicating vector containing a POI-encoding nucleic acid sequence, or by recombinant integration of one or more copies of a POI-encoding nucleic acid sequence into the genome of a host cell; Produced by recombinant modification of one or more regulatory sequences that control the expression of the encoded gene (eg, promoter sequence). In some cases, the term POI as used herein also refers to any metabolic product by the host cell that is mediated by the recombinantly expressed protein.

The term "sequence identity" of a variant, homolog, or ortholog indicates the degree of identity of two or more sequences as compared to a parent nucleotide or amino acid sequence. Two or more amino acid sequences may have, to some extent, up to 100%, the same or conserved amino acid residues at corresponding positions. Two or more nucleotide sequences may have, to some extent, up to 100%, identical or conserved base pairs at corresponding positions.

Sequence similarity searches are an effective and reliable strategy for identifying homologues with excess (eg, at least 50%) sequence identity. Frequently used sequence similarity search tools are, for example, BLAST, FASTA, and HMMER.

Sequence similarity searches can identify such homologous proteins or genes by detecting excess similarities and statistically significant similarities that reflect common ancestry. Homologs may include orthologs, which are understood herein as the same protein in different organisms (eg, variants of such a protein in different organisms or species).

With respect to amino acid sequences, homologs, and orthologs described herein, "percent (%) amino acid sequence identity" refers to (as part of the sequence identity) It is defined as the percentage of amino acid residues in a candidate sequence that are identical to amino acid residues in a particular polypeptide sequence after achieving the maximum percent sequence identity (without considering any conservative substitutions). Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms necessary to achieve maximal alignment over the entire length of the sequences being compared.

For purposes described herein, sequence identity between two amino acid sequences is determined using the NCBI BLAST program version BLASTP 2.8.1 with the following exemplary parameters: Program: blastp, word size: 6, expected value: 10, hitlist size: 100, gap cost: 11.1, matrix: BLOSUM62, filter string: F, composition adjustment: conditional composition score matrix adjustment.

The EMBOSS Needle web server (https://www.ebi.ac.uk/Tools/psa/emboss_needle/) uses the default settings (matrix: EBLOSUM62, Gap open: 10, gap extend: 0.5, end gap penalty: false, end gap open: 10, end gap extend: 0.5). EMBOSS Needle uses the Needleman-Wunsch alignment algorithm to find the optimal alignment of two input sequences (including gaps) and writes the optimal global sequence alignment to a file.

For example, "percent (%) identity" with respect to nucleotide sequences of promoters or genes is defined by aligning the sequences, introducing gaps if necessary (taking into account any conservative substitutions as part of the sequence identity). It is defined as the percentage of nucleotides in a candidate DNA sequence that are identical to nucleotides in the DNA sequence after achieving the maximum percent sequence identity (without). Alignments for the purpose of determining percent nucleotide sequence identity can be accomplished in a variety of ways within the art, eg, using publicly available computer software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms necessary to achieve maximal alignment over the entire length of the sequences being compared.

For purposes described herein (unless otherwise indicated), sequence identity between two amino acid sequences was determined using the NCBI BLAST program version BLASTN 2.8.1 using the following exemplary parameters. Determined: Program: blastn, word size: 11, expected threshold: 10, hit list size: 100, gap cost: 5.2, match/mismatch score: 2, -3, filter string: low complexity region, Mark only lookup tables.

As used herein with respect to a POI, the term "isolated" or "isolated" refers to the presence of a POI in its natural form, such as in a "purified" or "substantially pure" form Refers to such compounds that are sufficiently separated from the environment with which they may be associated (particularly cell culture supernatants). However, "isolated" does not necessarily refer to an artificial or synthetic mixture with other compounds or materials, or of impurities that may be present, e.g. due to incomplete purification, but do not interfere with the essential activity. This does not mean excluding its existence. An isolated compound may be further formulated to produce a preparation thereof, or may be further isolated for practical purposes. For example, the POI can be mixed with a pharmaceutically acceptable carrier or excipient when used in diagnosis or therapy.

As used herein, the term "purified" refers to at least 50% (mol/mol), preferably at least 60%, 70%, 80%, 90%, or 95% of a compound (e.g. POI). Purity is determined by methods appropriate to the compound (eg, chromatographic methods, polyacrylamide gel electrophoresis, HPLC analysis, etc.). Isolated and purified POIs described herein can be obtained by purifying cell culture supernatants to reduce impurities.

Isolation and purification methods for obtaining recombinant polypeptide or protein products include methods that exploit differences in solubility (e.g., salting out and solvent precipitation), methods that exploit differences in molecular weight (e.g., ultrafiltration and methods that utilize differences in charge (e.g., ion-exchange chromatography), methods that utilize specific affinity (e.g., affinity chromatography), and methods that utilize differences in hydrophobicity (e.g., reverse Phase-high liquid chromatography), and methods that utilize differences in isoelectric points (eg, isoelectric focusing) can be used.

The following standard methods are preferred: cell (debris) separation and washing by microfiltration or tangential flow filtration (TFF) or centrifugation, POI purification by precipitation or heat treatment, POI activation by enzymatic digestion, ion exchange (IEX), Hydrophobic interaction chromatography (HIC), affinity chromatography, size exclusion (SEC) or HPLC chromatography, POI precipitation, concentration and washing (eg ultrafiltration step).

A highly purified product is essentially free of contaminant proteins and preferably has a purity of at least 90%, more preferably at least 95%, or even at least 98% and up to 100%. Purified products can be obtained by purification of cell culture supernatants or otherwise from cell debris.

Isolated and purified POIs can be identified by conventional methods such as Western blot, HPLC, activity assay, or ELISA.

The term "recombinant" as used herein shall mean prepared by or the result of genetic manipulation. A "recombinant cell" or "recombinant host cell" is understood herein as a cell or host cell that has been genetically engineered or modified to contain a nucleic acid sequence that is not natural to the cell. A recombinant host may be engineered to delete and/or inactivate one or more nucleotides or nucleotide sequences, particularly with nucleotide sequences foreign to the host containing the recombinant nucleic acid sequence. It may specifically include an expression vector or a cloning vector. Recombinant proteins are produced by expressing the respective recombinant nucleic acids in a host. As used herein, the term "recombinant" with respect to a POI refers to a POI prepared, expressed, produced by recombinant means, such as a POI isolated from a host cell that has been transformed or transfected to express the POI. or isolated POI. According to the present invention, conventional molecular biology, microbiology, and recombinant DNA techniques within the art can be used. Such techniques are well explained in the literature. See, eg, Maniatis, Fritsch & Sambrook, "Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, (1982)."

Certain recombinant host cells are "engineered" host cells, understood as host cells that have been manipulated using genetic manipulation, ie, by human intervention. When a host cell is engineered to express, co-express, or overexpress a given gene or its respective protein, the host cell is The gene or protein is engineered to have the ability to express the gene or protein, respectively, as compared to a host cell that has not been engineered to express, co-express, or overexpress such gene or protein. As described herein, the yield of protein of interest (POI) can be determined without co-expressing or overexpressing a TIF-encoding polynucleotide or without co-expressing or overexpressing a TIF-encoding polynucleotide. The TIFs described herein can be increased by co-expression or overexpression when compared to the same cells expressing the same POI under the same culture conditions without being engineered to express it.

Surprisingly, it was found that overexpression of TIF, which is part of the mRNP and not a subunit of eIF3, led to increased production and secretion of several recombinant POIs.

According to certain examples described herein, overexpression of TIF enhanced the translational capacity of engineered cells and also correlated with higher levels of POI and endogenous transcripts.

More surprisingly, overexpression of single TIFs, such as eIF4A, eIF4G, eIF4E, PAB1, and RLI1, as well as their combinations, in different culture modes (screening, fed-batch, and continuous culture) significantly reduced POI production. Yield increased.

The foregoing description will be more fully understood by reference to the following examples. However, such examples are merely representative of ways of implementing one or more embodiments of the invention and should not be construed as limiting the scope of the invention.

Example 1: Construction of translation factor (TIF) overexpression strain a) Host strain and expression vector:
P. C. pastoris strain CBS7435 or CBS2612 (CBS-KNAW Fungal Biodiversity Center, Central Bureau Schimmelcultures, Utrecht, The Netherlands ds) was used as the background strain. To assess the effects of overexpression of translation factors, different secreted model proteins were used as reporters. For this purpose, CBS2612_PG1-3_vHH#4 (described in WO2020/144313A1) was _used for expression. To generate additional host strains, the expression cassette of _{PG1-3_HSA} was transformed into CBS2612 and the expression cassette of _{PG1-3_vHH} was transformed into CBS7435 as described in WO2020/144313A1. MutS P _AOX1 -vHH (Zavec et al. 2020, Biotechnol Bioeng. 117(5):1394-1405) was used to evaluate the effect in methanol conditions.

b) Generation of TIF overexpression vectors Targets were overexpressed by homologous recombination of the respective expression cassettes into the host strain. Plasmids for this were generated by using two different cloning strategies. Selected target translation factors are shown in Tables 2 and 7.

P. by pPuzzle-based expression. Construction and selection of strains overexpressing P. pastoris translation factors The pPM2aK21 plasmid, which is a derivative of the pPuzzle_ZeoR vector backbone described in WO2008/128701A2, was used. This includes the AOX terminator sequence (for integration into the natural AOX1 terminator locus), the E. coli origin of replication (pUC19), the antibiotic resistance cassette MCS (kanamycin and kanMX) conferring resistance to G418), an expression cassette for the gene of interest (GOI) consisting of the GAP promoter, multiple cloning site (MCS), and the S. cerevisiae CYC1 transcription terminator. Selected overexpressed genes were amplified from the start codon to the stop codon by PCR (Q5® High Fidelity DNA Polymerase, New England Biolabs) using the primers shown in Table 8. The sequence was cloned into the MCS of the pPM2a expression vector using the two restriction enzymes SbfI and SfiI. Gene sequences were verified by Sanger sequencing.

P. by GoldenPiCS system. Construction and selection of strains overexpressing P. pastoris translation factors Genes selected for overexpression are amplified from the start codon to the stop codon by PCR (Q5® High Fidelity DNA Polymerase, New England Biolabs) or It was split into several pieces. The GoldenPiCS system (Prielhofer et al. 2017. BMC Systems Biol. 11, 123, doi: 10.1186/s12918-017-0492-3) requires the introduction of silent mutations in several coding sequences. This was done by amplifying several fragments from one coding sequence by using the primers in Tables 3 and 9. Alternatively, gBlocks were obtained (Integrated DNA Technology IDT). P. Genomic DNA from P. pastoris strain CBS2612 was used as a PCR template. The obtained fragment was amplified with the primers shown in Tables 3 and 9, and then introduced into BB1 of the GoldenPiCS system by using the restriction enzyme BsaI. The GoldenPiCS system is composed of skeletons BB1, BB2, and BB3. The assembled BB1 carrying the respective coding sequences were combined with the promoter and terminator regions in BB2 and then further processed to generate the required BB3 integration plasmids as described in Prielhofer et al. (2017). did. All promoters and terminators used to assemble the expression cassettes in the BB2 or BB3 scaffolds are described in Prielhofer et al. (2017). The BB3rN used contains the 5'-RGI1 genomic integration region and a NatMX selection marker cassette for selection against noseothricin. All plasmids contain the E. coli origin of replication (pUC19).

c) Generation of TIF overexpression transformants The plasmid was linearized using the AscI restriction enzyme and then as described by Gasser et al. 2013 (Future Microbiol. 8(2):191-208). pastoris was electroporated. Positive transformants were tested on YPD plates (per liter: 10 g yeast extract, 20 g peptone, 20 g glucose, 20 g agar) containing 500 μg/mL ⁻¹ G418 or 100 μg/mL ⁻¹ noseothricin. made a selection. Colony PCR was used to confirm that the transformed plasmid was at the correct locus. For this, the genomic DNA is P. pastoris colonies were obtained by cooking them in 0.04 M NaOH, which were applied directly to PCR using appropriate primers.

d) Determination of gene copy number (GCN) of overexpression targets Expression intensity is often determined by P. pastoris genome. Therefore, the gene copy number of each of the overexpressed targets was determined. Genomic DNA was isolated using the Wizard® Genomic DNA Purification Kit (Promega Corporation, Cat. No. A1120). Quantitative real-time PCR (qPCR) was then used to determine gene copy number. The Blue S'Green qPCR kit (Biozym) was used for this. Blue S'Green qPCR master mix was mixed with primers and samples and applied for real-time analysis in a real-time PCR cycler (Rotor Gene, Qiagen). Table 1 shows the list of primers used. All samples were analyzed in triplicate. Rotor Gene software was used for data analysis. The ACT1 gene was used as a calibrator. GCN was determined by using the _PGAP primer for single overexpression constructs (see Example 4) and the TIF2 primer for combined overexpression constructs (see Example 5). Tables 10 and 12 show the results, respectively. The selected overexpression target was P. As an endogenous gene of P. pastoris, a GCN of 2 indicates successful integration of one additional gene copy, one of the genes being the original gene and the second being the overexpression cassette.

Example 2: Analysis of the effect of TIF overexpression on recombinant protein production To determine the effect of overexpression of translation factors on the secretion of recombinant proteins, engineered overexpression strains were engineered to use the pG promoter for GOI. Suitable conditions such as glucose-limiting conditions in the case of a promoter derived from the methanol utilization pathway (Prielhofer et al. 2013. Microb Cell Fact 12,5) or methanol induction in the case of a promoter derived from the methanol utilization pathway (Gasser et al. 2015. Microb. Cell Fact 14:196) The cells were cultured under suitable screening conditions. P. Engineering of P. pastoris host strains was accomplished by incubating either pPuzzle-based or GoldenPiCS BB3rN-based TIF expression vectors as described in Example 1. This was done by integrating into the S. pastoris genome. Then, the manipulated P. P. pastoris strains were cultured on a small scale (screening procedure), thereby simulating fed-batch culture. The recombinant protein secreted into the supernatant was quantified and the titers and yields of the different engineered strains were compared with the parental host strain.

_Medium : _Synthetic screening medium ASMv6 (per liter): 6.30g ( _NH4 ) _2HPO4 , 0.8g ( _NH4 ) _2SO4 , 0.49 _MgSO4 * _7H2O , 2.64g KCl, 0 .0535g _CaCl2 * _2H2O , 22.0g citric acid monohydrate, 1470μL PTM ₀ trace salt stock solution, 20mL _NH4OH (25%), 4mL biotin (0.1g/L ^-1 ). Solid KOH was added to set the pH to 6.4-6.6.

PTM ₀ trace salt stock solution (per liter): 5.0ml H ₂ SO ₄ (95-98%), 65.0g FeSO ₄ *7H ₂ O, 20g ZnCl ₂ , 6.00g CuSO ₄ *5H ₂ O, _3.36g MnSO4 * _H2O , 0.82g CoCl2 _{* 6H2O} , _{0.20g Na2MoO4 *} 2H2O, 0.08g NaI, _{0.02g H3BO3}

a) Engineered P.I. with GOI expression under the control of the pG promoter. Screening of P. pastoris strains For screening of model protein secretion, a single colony incorporating the PCR-verified gene at the correct locus was harvested in 2 mL of liquid YPG medium (per liter): 20 g peptone, 10 g yeast extract, 12.6 g 100% glycerin, pH 7.4-7.6) containing 50 μg/mL ⁻¹ Zeocin and 500 μg/mL ⁻¹ G418 or 100 μg/mL ⁻¹ Norseothricin (as applicable). Additionally, host strains were grown in quadruplicate on each plate for comparison. This preculture was grown at 280 rpm, 24 DWP for approximately 24 hours at 25°C. The preculture was then used to inoculate 2 mL of synthetic screening medium ASMv6 at a starting OD ₆₀₀ of 8. The medium contained 50 g/L ⁻¹ polysaccharide (EnPump200 polysaccharide, Enpresso) and 0.4% glucose-releasing enzyme (Reagent A, Enpresso) as carbon source. Culture conditions were similar to preculture conditions. After 48 hours, 1 mL of cell suspension was transferred to a pre-weighed 1.5 mL centrifuge tube and centrifuged at 16,000 g for 5 minutes at room temperature. The supernatant was carefully transferred to a new vial and stored at -20°C until further use. The centrifuge tube containing the pellet was reweighed to determine wet cell weight (WCW). Quantification of recombinant secreted proteins in the supernatant was performed by microfluidic capillary electrophoresis as described below.

b) An engineered P. elegans expressing GOI under the control of a methanol-inducible promoter. Screening for P. pastoris strains 2 mL YPD medium (per liter): 20 g peptone, 10 g yeast extract, 22 g D(+)-glucose monohydrate, pH 7.4-7.6) (50 μg/mL ⁻¹ Zeocin and ^{P. _} A single colony of P. pastoris clone was inoculated and grown overnight at 25° C. at 280 rpm and 24 DWP. The preculture was then used to inoculate 2 mL of synthetic screening medium ASMv6 at a starting OD ₆₀₀ of 8. The medium contained 25 g/L ⁻¹ polysaccharide (EnPump200 polysaccharide, Enpresso) and 0.35% glucose-releasing enzyme (Reagent A, Enpresso) as carbon source. These cultures were incubated at 25° C. for 48 hours at 280 rpm and 24 DWP. After the first 3 hours, cells were fed with 10 μL (0.5%) of pure methanol. Cells were then refed with 20 μL (1%) of pure methanol after 19, 27, and 43 hours of culture time. After 48 hours, 1 mL of cell suspension was transferred to a pre-weighed 1.5 mL centrifuge tube and centrifuged at 16,000 g for 5 minutes at room temperature. The supernatant was carefully transferred to a new vial and stored at -20°C until further use. The centrifuge tube containing the pellet was reweighed to determine wet cell weight (WCW). Quantification of recombinant secreted proteins in the supernatant was performed by microfluidic capillary electrophoresis, as described below.

c) Quantification of secreted recombinant proteins by microfluidic capillary electrophoresis (mCE) A “LabChip GX/GXII system” (PerkinElmer) was used for quantitative analysis of secreted protein titers in culture supernatants. The consumables "Protein Expression Lab Chip" (760499, PerkinElmer) and "Protein Expression Reagent Kit" (CLS960008, PerkinElmer) were used. Chip and sample preparation were performed according to the manufacturer's recommendations. A brief description of the procedure is provided below.

Chip preparation: After the reagents reached room temperature, 520 μL and 280 μL of protein expression gel matrix were transferred to a spin filter. 20 μL of protein expression dye solution was added to a spin filter containing 520 μL of gel matrix. After briefly vortexing the spin filters containing dye upside down, both spin filters were centrifuged at 9300 g for 10 min. To wash the chip, 120 μL of Milli-Q® water was added to all active chip wells and the chip was subjected to an instrument cleaning program. After two additional rinse steps with Milli-Q® water, the remaining fluid was aspirated completely and the appropriate amounts of filtered gel matrix solution as well as protein expression low molecular weight marker solution were added to the appropriate chip wells.

Sample and ladder preparation: For sample preparation, 6 μL of sample was mixed with 21 μL of sample buffer in a 96 microtiter plate. Samples were denatured at 100°C for 5 minutes and centrifuged at 1,200g for 2 minutes. Then 105 μL of Milli-Q® water was added. Sample solutions were briefly mixed by pipetting and centrifuged again at 1,200 g for 2 min before measurement. To prepare the ladder, 12 μL of protein expression ladder was denatured at 100° C. for 5 minutes in a PCR tube. Then, 120 μL of Milli-Q® water was added, the ladder solution was briefly vortexed, and the tube was spun for 15 seconds in a small microcentrifuge to begin the measurement.

Quantification was performed by comparison to BSA standards using LabChip software provided by the manufacturer.

Example 3: P. Effect of overexpression of translation initiation factor 3 (eIF3) subunit on secretion of recombinant proteins in P. pastoris.
First, a subunit of translation initiation factor 3 (eIF3) was overexpressed. This factor consists of six subunits in yeast, which were overexpressed by themselves and in different combinations. For overexpression, the eIF3 subunit was cloned into the GoldenPiCS vector and transformed into host strain CBS2612 _PG1-3 vHH#4 as described in Example 1. The engineered strains were then screened and yields compared to the host strain as described in Example 2.

a) Overexpression of a single subunit of eIF3.
The subunits of eIF3 shown in Table 2 were amplified using the primers shown in Table 3 and cloned into host strain CBS2612 _PG1-3 vHH#4 as described in Example 1. No overexpression vector was obtained for eIF3a.

Table 4 shows the results of single overexpression of eIF3 subunit. Each target gene was overexpressed with a different promoter and approximately 10-fold overexpression was achieved. These approximate overexpression intensities are shown in the OE column and were calculated as described in Example 4a. Screening results are expressed as fold change in vHH yield compared to host strain. The results in Table 4 clearly show that overexpression of a single eIF3 subunit does not affect the secretion of recombinant proteins in Pichia pastoris (increased in vHH yield between engineered strains and controls). All fold changes were approximately 1, meaning there was no difference in protein production between the engineered strain and the parental control strain). Roobol et al. (Metabolic Engineering 2020, 59:98-105) reported increased proliferation rate and increased protein synthesis capacity upon transient and stable overexpression of eIF3i and eIF3v subunits in mammalian HEK cells and CHO cell lines. This was unexpected, as it had been reported that

a) Combination overexpression of eIF3 subunits.
Next, different combinations of eIF3 subunits were selected for overexpression as listed in Table 5. Cloning and transformation were performed as described in Example 1 and the resulting strains were screened as described in Example 2. As described in Example 4a, the promoter was chosen to keep the intracellular transcript concentration ratio the same as the native strain. The OE column shows the calculated overexpression intensity.

Table 6 shows the fold change in vHH yield compared to host strain. In the screen, even combinations of overexpressing several subunits of eIF3 did not increase vHH production. As in Example 3a, the fold changes are all approximately 1, meaning that there is no significant increase in recombinant protein secretion when eIF3 subunits are overexpressed alone or in combination.

Example 4: Effect of overexpression of a single TIF of mRNP on recombinant protein production Translation factors that act on translation initiation and are part of the mRNP and closed-loop complexes: eIF4A, eIF4E, eIF4G, PAB1, and RLI1 ( Table 7) was selected for overexpression purposes and an overexpression vector was constructed as in Example 1b using the primers shown in Tables 8 and 9. _{CBS2612_PG1-3_vHH} #4 (described in WO2020/144313A1) was used as the parent host strain and transformed with the single TIF overexpression vector described in Example 1.

a) Generation of single OE vector and determination of OE strength

For all single TIF overexpressions described, the strong constitutive pGAP promoter was used and the TIF expression cassette was integrated into either the 5'-RGI1 locus or the AOX1 transcription terminator. Rebnegger et al. 2014. Biotech J. 9(4):511-25, the expected degree of overexpression by the selected promoters was calculated. The "OE" column in Table 10 shows the estimated increase in expression intensity compared to the parent strain.

Table 10 also shows the measured GCN of the selected clones and the results of the screening procedure. All clones shown in Table 10 each contained one additional copy of the TIF gene (denoted as GCN=2).

b) Effect of single TIF overexpression on recombinant protein production using vHH under the control of pG1-3 as reporter Strains were cultured as described in Example 2a and after 48 h of culture. The titer of secreted vHH was determined by mCE (Example 2c). For each clone, the titer (mg vHH/L ⁻¹ ), WCW (g/L ⁻¹ ), and biomass-specific product yield (mg vHH/g ⁻¹ WCW) were calculated, and then one factor was averaged over all clones overexpressing as well as the replication of the parental strain. Fold change in titer (FC) and yield were determined compared to the average of the parental host strain grown on the same 24-DWP.

Unexpectedly, even single overexpression of selected TIFs of mRNPs clearly increased recombinant protein production and secretion (Table 10). The highest improvement was seen with TIF4632 (eIF4G) and PAB1 overexpression, both of which increased secretion of recombinant protein approximately 2.0-fold.

Example 5: Effect of overexpression of TIF combinations of mRNPs on secretion of recombinant proteins.
In order to analyze whether there is an effect of overexpression of some translation initiation factors in the complex, we selected different combinations of translation factors tested in Example 4 and investigated their effects on the production of recombinant proteins. compared. This combination operation was performed using the GoldenPiCS toolbox as described in Example 1b. The resulting plasmid was transformed into host strain CBS2612 _PG1-3 vHH#4. Then, the manipulated P. P. pastoris strains were cultured on a small scale as described in Example 2. The proteins secreted into the supernatant were measured as in Example 2c and the titers and yields of the different engineered strains were compared to the parental host.

a) Combinations generated for overexpression of translation initiation factors.
Overexpression of different combinations listed in Table 11 was tested. The promoters described were selected to overexpress each gene approximately 10-fold. This was done to balance the concentration ratio of transcripts of different target genes in the cells. However, as seen in Table 10, stronger or weaker overexpression can also be selected, still leading to increased production of recombinant protein.

a) Effect of combined overexpression on secretion of recombinant proteins.
Engineered strains were screened and data analyzed as described in Example 2.

Table 12 shows the effect of overexpression of different TIF combinations on recombinant protein production. All clones shown in Table 12 were verified to have the overexpression cassette inserted only once. This means that they exhibit a GCN of 2. The fold change in vHH yield is shown compared to host strain CBS2612 P _G1-3 vHH#4. All combinations showed increased secretion of recombinant vHH compared to the parent, but the combination of C3 and C13 clearly shows the greatest effect. C3a and C3b are P. contains different versions of TIF2 with different lengths according to different annotations in the P. pastoris genome sequence. Independently of the TIF2 version, both combinations increased vHH yield more than 2-fold. C3b increased vHH yield by 2.5 times.

b) Comparison of C3b overexpression to secretion of recombinant protein in different background strains.
To compare the effects of different background strains, the P _{G1-3_vHH} expression cassette was transformed into CBS7435 as described in Example 1a. Construct C3b was then integrated into the genome of the resulting CBS7435 P _G1-3 vHH producing host strain as described in Example 1c. The effects of C3b overexpression were then screened as described in Example 2a, and secreted vHH titers were determined as described in Example 2c. For comparison, nine different clones of CBS7435 P _G1-3 vHH C3b were screened and compared to the biological quadruplicate of host strain CBS7435 P _G1-3 vHH.

Table 13 shows the fold change in vHH yield of C3b overexpression in host strain CBS7435 P _G1-3 vHH. These results indicate that a strong beneficial effect on the secretion of recombinant vHH is seen in all strains with C3b overexpression, independent of background strain selection.

c) Effect of TIF overexpression on methanol-inducible recombinant protein secretion.
To determine the effect of C3b overexpression on methanol-inducible recombinant protein secretion, CBS7435 MutS containing the pAOX1-vHH expression cassette (Zavec et al. 2020, Biotechnol Bioeng. 117(5):1394-1405) was It was used as a host strain for C3b overexpression. Methanol shots for _PAOX1 induction were used to screen strains as described in Example 2b and protein titers were determined as described in Example 2c.

Screening using 10 C3b overexpressing clones showed an average 1.39±0.05-fold increase in vHH yield compared to the parent strain. This confirms that increased secretion of recombinant proteins can be achieved with TIF overexpression, regardless of the applied carbon source or promoter system.

Example 6: Characterization of the effects of overexpression of translation initiation factors on cellular processes.
To assess which cellular processes were affected by overexpression of translation initiation factors in addition to the observed differences in recombinant protein secretion, two different approaches were followed: on the one hand, gene transcription levels were Measurements were taken to determine potential effects on transcript abundance (Example 6a). On the other hand, P. Cellular translational activity was directly measured after setting up a puromycin-based method in P. pastoris (Example 6b).

a) Spike-in method for comparative measurement of transcript levels.
Strains were first cultured for 30 hours in the 24-DWP screening procedure as described in Example 2. This corresponds to a growth rate of approximately 0.025/h ^-1 at the time of harvest. 1 mL of culture was harvested and centrifuged at 16,000 g for 5 minutes at 4°C. The supernatant was discarded and the pellet was stored at -80°C until further use.

The pellets were dissolved in PBS and pelleted again according to WCW to have the same amount of yeast mass in each sample so that potential changes in transcript concentrations of common housekeeping genes could also be measured. Make it. Each pellet was then injected into 1 mL of S.I. cerevisiae S288c suspension (aliquot from a single shake flask culture). The resulting mixed P. pastoris-S. The S. cerevisiae pellet was used for RNA isolation.

For RNA isolation, 1 mL of TRI reagent (Sigma-Aldrich) and 500 μL of acid-washed glass beads were added to the cells and then disrupted for 40 s at a speed of 5.5 m/s in FastPrep-24 (mpbio). did. Then, 200 μL of chloroform was added. The samples were then shaken vigorously and then left at room temperature for 5-10 minutes. After centrifugation at 16,000 g for 10 min at 4 °C to promote phase separation, the upper colorless aqueous phase containing the RNA was transferred to a new tube and 500 μL of isopropanol was added to precipitate the RNA. After 10 minutes of incubation, samples were centrifuged at 16,000 g for 10 minutes at 4°C and the supernatant was discarded. The RNA pellet was washed once with 70% ethanol, air dried, and resuspended in RNAse-free water.

To remove residual DNA, RNA samples were treated with the DNA-free™ kit (Ambion) according to the manufacturer's manual. RNA quality, purity, and concentration were then analyzed by gel electrophoresis and spectrophotometric analysis using a NanoDrop2000 (Thermo Scientific).

cDNA synthesis was performed using the Biozym cDNA synthesis kit according to the manufacturer's manual. Briefly, 1 μg of total RNA was added to a master mix containing reverse transcriptase, dNTPs, RNase inhibitors, and synthesis buffer. Oligo d(T)23VN (NEB) was used as priming. Incubation of the reaction mixture was carried out for 45 minutes at 55°C. Enzyme inactivation was then achieved by incubating the reaction mixture at 99°C for 5 minutes.

P. For quantitative real-time PCR (qPCR) of P. pastoris ACT1, primers specific for TDH3 and vHH were used (see Table 14). Normalization is performed by S. cerevisiae ACT1 (see Table 14). Transcript levels of engineered strains were compared to those of the host strain. Both sets of ACT1 primers were tested and verified to bind only to the desired organism's cDNA. For qPCR, 1 μL of cDNA, water, and primers were mixed with Blue S'Green qPCR master mix (Biozym) and analyzed in a real-time PCR cycler (Rotor-Gene, Qiagen). All samples were technically measured in triplicate. Data analysis was performed with Rotor-Gene software using comparative quantification (QC) methods.

b) Effect of TIF overexpression on transcript abundance The TIF overexpression strains shown in Table 15 and the host strain CBS2612 _PG1-3 vHH#4 were subjected to the 24-DWP screening procedure as described in Example 2a. The cells were cultured for 30 hours. Transcript abundances of the two endogenous genes and the recombinant GOI were determined as described in Example 6a.

Table 15 shows the obtained results of transcription level measurements. Measurements show that vHH transcription levels are strongly affected by TIF overexpression. In Examples 4 and 5, particularly high values are seen for the overexpression combinations that already showed higher secretion of recombinant protein. The highest transcript levels were found in strains overexpressing C3b. This overexpression increased the transcription level of vHH by 5.5-fold. Surprisingly, TDH3 expression also appears to be increased in all overexpression lines, whereas ACT1 expression appears to be increased especially in the combined overexpression lines. Increased transcript levels of both housekeeping genes ACT1 and TDH3 indicate an increase in all transcripts within the cell. These results indicate that overexpression of TIF has a positive and/or stabilizing effect on cellular mRNA levels, which may be one factor leading to increased productivity.

c) Measurement of global translational activity.
Measurement of global translational activity with O-propargyl-labeled puromycin was performed as described by Nagelreiter et al. after optimizing the procedure for use in yeast cells. 2018. It was performed in the same manner as Biotechnol J 13, e1700492. Briefly, cells from the same culture as Example 6a were pipetted into a 96-well microtiter plate in 90 μL of “incubation solution” at a final OD ₆₀₀ of 0.4. The "incubation solution _" was 0.6mM dissolved _in 10% DMSO and PBS (2mM _KH2PO4 , 10mM _Na2HPO4.2H2O , 2.7mM g KCl, 8mM _NaCl , pH 7.4). It consisted of ASMv6 medium (see Example 2) containing O-propargyl puromycin (Jena Bioscience, NU-931-05) and 1.5 g/L ⁻¹ imipramine. The suspension was incubated on a shaker for 2 hours at 25°C, transferred into ice-cold Eppendorf tubes, and centrifuged at 16,000 g for 5 minutes at 4°C. After washing the pelleted cells with 120 μL of PBS, the pelleted cells were again fixed with 1 mL of ice-cold 70% ethanol. These fixed samples were stored at 4°C for 1 day to 2 weeks.

For click chemistry, fixed samples were collected by centrifugation at 16,000 g for 5 min at 4°C. The pellet was transferred to a 96-well microtiter plate and washed with 100 μL of “click chemistry buffer” (115 mM Tris/HCl pH=8.5, 0.1% TritonX-100). The samples were then incubated in "Click Chemistry Mix" (101 mM Click-It Click chemistry buffer, 1.9 mM CuSO4, 1.9 mg/mL ascorbic acid, 20 μM Alexa Fluor™ 488 azide (Invitrogen)) for 30 minutes at room temperature. Incubated. Cells were then harvested as before, washed in 150 μL of PBS, and lysed in 150 μL of fresh PBS. To measure the resulting fluorescence intensity, cells were analyzed by flow cytometry at an excitation wavelength of 488 nm and an emission wavelength of 525 nm. For each sample, 40,000 events were measured. For data analysis, the geometric mean of each sample was used and the blank (cells treated without O-propargyl puromycin addition) was subtracted from each.

d) Effect of overexpression of TIF on global cellular translation. Table 16 shows the fold change in fluorescence values obtained and therefore the average overall translational activity compared to host strain CBS2612 _PG1-3 vHH#4. The fold change is shown. The same clones shown in Table 15 were used to measure translation activity. Translation activity was determined as described in Example 6c.

Table 16 clearly shows that overexpression of a single translation initiation factor resulted in improved translational activity in each cell. Overexpression of selected TIF combinations shows an even stronger increase in translational activity. This is also reflected by the increased secretion of recombinant proteins observed in these strains (Examples 4 and 5). The highest translational activity, 2.3 times higher than the host strain, can be achieved by overexpressing C3b. These results indicate that overexpression of selected translation initiation factors or combinations thereof increases not only translation of specific proteins, such as recombinant proteins, but also overall cellular translational activity. Surprisingly, there was a clear correlation between the improvement in vHH yield (Tables 10 and 12) and relative translational activity (correlation coefficient R ² =0.84), which could be attributed to the shows that formation is the rate-limiting step in the production of recombinant proteins.

Example 7: Effect of overexpression of translation factors in fed-batch culture.
To further validate the observations made in the screen, fed-batch cultures similar to standard production processes were performed using the selected overexpression targets.

a) Effect of TIF overexpression on vHH production in fed-batch culture:
In this example, _{CBS2612_PG1-3_vHH} #4 overexpressing either C3b or RLI1 was selected for culture. These strains showed strong beneficial effects on secretion of recombinant proteins in screens (Examples 4, 5, and 9). For fed-batch culture, using the same medium but applying different feeding profiles, the following calculated growth rates were obtained at each sampling point (Table 17). Media composition can be found below.

Culture medium:
PTM ₀ trace salt stock solution (per liter):
5.0 mL H ₂ SO ₄ (95-98%), 65.0 g FeSO ₄ *7H ₂ O, 20 g ZnCl ₂ , 6.00 g CuSO ₄ *5H ₂ O, 3.0 g MnSO ₄ *H ₂ O, 0. 5g _CoCl2 * _6H2O , _{0.20g Na2MoO4} * _2H2O , 0.08g NaI _, 0.02g _H3BO3

Glycerol batch medium (content per liter):
2g citric acid monohydrate ( _C6H8O7 * _H2O ) _, 45g glycerol, 12.6g ( _NH4 ) _2HPO4 , _{0.5g MgSO4} * _7H2O , 0.9g _KCl , 0 .022 g CaCl ₂ *2H ₂ O, 13.2 mL biotin stock solution (0.1 g/L ⁻¹ ), and 4.6 mL PTM0 trace salt stock solution. (Conc.) HCl was added and the pH was set to 5.

Glucose feeding medium (content per liter):
495g glucose monohydrate, 4.6g _MgSO4 * _7H2O , 8.4g KCl, 0.28g CaCl2* _2H2O , 23.6mL biotin stock solution (0.1g/L ^-1 ), and ₁₀ .1 mL PTM0 trace salt stock solution.

b) Fed-batch culture with linear feeding with a minimum growth rate reaching 0.04/h ^-1 .
Fed-batch culture was carried out using the host strain CBS2612 _PG1-3 vHH#4 and the corresponding overexpression strains CBS2612 _PG1-3 vHH C3b#13, CBS2612 _PG1-3 vHH C3b#16, and CBS2612 _PG1-3 vHH _PGAP. RLI1 #4 was used in a 1 L benchtop bioreactor (SR0700ODLS; Germany; reactor runs #A-B) or a 1.8L benchtop bioreactor (SR15000ODLS; Dasgip, Germany; reactor run #C). For pre-culture, 100 mL of YPG medium containing 50 μg/mL ⁻¹ zeocin and 100 μg/mL ⁻¹ notheothricin (if applicable) in a 1 L shake flask was inoculated with 1.0 mL cryostoc and incubated at 180 rpm. , and incubated at 25°C for approximately 24 hours. Batch cultures were operated in a working volume of 0.5 L and inoculated at a starting OD ₆₀₀ of 1.5. The media composition of the glycerol batch is shown above. Throughout the process, the temperature was controlled at 30 °C and the DO was maintained at 30% and 12.5% by automatic adjustment of stirrer speed (400-1200 rpm) and air flow rate (9.5-30 sL/hr ^-1 ). The pH was adjusted to 5.0 by automatic addition of NH ₄ OH. After a sudden spike in DO, marking the end of batch (BE), a rapid initial growth rate (μ) is followed by a linearly increasing glucose supply (medium composition detailed above) leading to an expansion phase of decreasing μ. ) was applied. The linear increase in feed was set to follow the following formula: F [mL/hr− ¹ ]=0.1431*t+2.0499. In order to confirm the obtained results, the same fed-batch culture was performed twice.

Yeast dry mass (YDM) and secreted recombinant protein were analyzed at various times throughout the process (shown in Table 18). For YDM analysis, 1 mL of culture broth was transferred to a 2 mL centrifuge tube that had been previously dried (at least 24 hours at 105° C.) and preweighed. After centrifugation at 16,000g for 5 minutes at 4°C, the supernatant was carefully transferred to a new vial and stored at -20°C until further use. Cell pellets were washed twice with 0.1 M HCl and dried at 105° C. for at least 24 hours before being weighed again.

Supernatants were analyzed by microfluidic capillary electrophoresis (GXII, Perkin-Elmer) as described in Example 2c.

It can be seen in Table 18 that overexpression of TIF did not affect the biomass concentration in fed-batch culture. In contrast, product titer and yield increased over the course of the fed-batch compared to the parent control. In particular, at later time points (F20 and F46) a clear positive effect of RLI1 overexpression on product titer and yield was seen, exceeding the parent host strain by 2.2 times at the end of fermentation. Overexpression of C3b increased product yield and titer by an average of 2.7-fold.

c) Culture with constant feeding with a minimum growth rate of 0.02/h ^-1 .
Separate fed-batch cultures were grown with the host strain CBS2612 P _G1-3 vHH#4 and the corresponding overexpression strains CBS2612 P _G1-3 vHH C3#16 and CBS2612 P _G1- as described in Examples 7a and 7b. ₃ vHH _PGAP RLI1#4 was used. However, in this culture a different feed profile was chosen and a constant glucose feed was applied instead of the linear incremental glucose feed as described in Example 7b. A constant feed was maintained at 4 mL/hr ⁻¹ during the entire fed-batch culture. This rapidly reduced the initial growth rate, resulting in slower growth and longer culture periods. Sampling was performed as described above. In addition to YDM and supernatant, 1 mL of cell suspension was collected, pelleted, and frozen at -80°C for determination of transcript levels.

Table 19 also shows that with this feeding strategy, both RLI1 and C3b overexpression resulted in increased product titer and yield compared to the host strain while producing the same amount of YDM. show. As in Example 7b, overexpression of C3b proves to be highly beneficial for recombinant protein production, reaching an average of 4 times higher product yield and titer.

Taken together, this shows that overexpression of single TIFs or their combination has a strong positive effect on recombinant protein production, independent of the applied feeding strategy.

d) Effect of overexpression of translation factors on transcription levels in fed-batch.
To assess whether the effect of TIF overexpression on transcript abundance seen in Example 6b persists when cells are cultured in fed-batch, transcript levels of PpACT1, vHH, and TDH3 were Analyzed on samples from batch runs C041-C044 (Example 7b). The procedure was performed as described in Example 6a. As described above, transcription levels were determined in S. Normalized to S. cerevisiae ACT1. In addition, they were then normalized to the host strain, Reactor C041, at the corresponding sampling points. Table 20 shows the fold change in relative transcript levels of PpACT1, vHH, and TDH3.

Overexpression of RLI1 resulted in a slight increase in the transcript levels of the three genes analyzed (average of 1.2 for the two native P. pastoris genes at later time points F31 and F54), whereas overexpression of C3b produced up to 1.5-fold increase in PpACT1, up to 2.9-fold increase in vHH, and up to 2.2-fold increase in TDH3 (Table 20). Increased vHH transcript levels in C3b appear to correlate with increased titers seen in the same fed-batch cultures (Table 19). Elevated transcript levels of both housekeeping genes ACT1 and TDH3 indicate elevated levels of all transcripts in the cells, regardless of culture mode.

e) Fed-batch culture with methanol-inducible recombinant protein secretion.
Fed-batch cultivation of clones requiring methanol induction was performed according to standard processes and media for MutS strains as described in Zavec et al. (2020). For pre-culture, 100 mL of YPG medium containing 50 μg/mL ⁻¹ zeocin and 100 μg/mL ⁻¹ noseothricin (if applicable) in a 1 L shake flask was inoculated with 1.0 mL cryostoc; It was incubated for about 24 hours at 180 rpm and 25°C.

Batch cultures were operated with a working volume of 0.4 L of BSM medium (Mellitzer et al., 2014) and inoculated at a starting OD ₆₀₀ of 2.5. The temperature was controlled and maintained at 25° C., and the DO was maintained at 20% by automatic adjustment of stirrer speed (200-1250 rpm), air flow rate (9.5-50 sL/hr ⁻¹ ), and oxygen supplementation. The pH was adjusted to 5.0 by automatic addition of 25% _NH4OH . After a sudden spike in DO indicating end of batch (BE), the glycerol feed was started followed by the glycerol/methanol co-feed. Glycerol feeding (60% (w/w) + 12 mL/L PTM1) with linearly increasing (y=0.225x+1.95) glycerol feeding lasted for 8 hours. This was followed by an 18 hour co-feed of 60% glycerol and 100% methanol. For co-feeding, the 60% glycerol feed decreased linearly (y=3.75-0.111x) and the methanol feed increased linearly (y=0.028x+0.6). Finally, a linearly increasing methanol feed (y=0.028x+1.10) was applied for 72 hours with a methanol-only feed phase. Sampling, YDM determination, and protein quantification were performed as described in Example 7b.

Table 21 also shows that when using a methanol-based recombinant protein production strategy, overexpression of C3b results in increased product titer and yield compared to the host strain, and thus has a distinct beneficial effect. Show that it is effective. The increase is approximately 1.7-fold and begins immediately after the start of pure methanol feeding and continues until the end of the culture.

Example 8: Effect of TIF overexpression in chemostat culture.
Since continuous culture is attracting more attention in the field of biopharmaceutical production, the effect of C3b overexpression on the secretion of recombinant proteins was also analyzed at a fixed growth rate in chemostat culture. This method offers the possibility of continuous culture during the production process and allows tight control of the growth rate.

a) Effect of overexpression of C3b on recombinant protein secretion at a fixed growth rate in the chemostat.
Culture medium:
Chemostat trace element solution (per liter):
15g EDTA, _4.5g ZnSO4 * _7H2O , _1.03g MnCl2 _{* 4H2O} , 0.3 _CoClo2 * _6H2O , 0.3g _CuSO4 , 0.4g _Na2MoO4 * _2H2O , 4.5g _CaCl2 * _2H2O , 3g _FeSO4 * _7H2O , _{1g H3BO3} , 0.1 KI.
EDTA and ZnSO ₄ *7H ₂ O were dissolved in H ₂ O and the pH was set to 6 with solid NaOH, then the other salts were dissolved one by one. The pH was then set to 4 with solid NaOH and concentrated HCl.

Chemostat glucose medium (per liter) (to achieve YDM of 10 g/L ⁻¹ ):
22 g glucose monohydrate, 10 g (NH ₄ ) ₂ SO ₄ , 6 g KH ₂ PO ₄ , 1 MgSO ₄ *7H ₂ O, 0.5 g Pluronic® PE6100, 3 mL Trace element solution for chemostats, 1.6 mL Biotin stock solution (0.1 g/L ⁻¹ ).

The pH was set to 5 by adding solid KOH.

For chemostats, strain CBS2612 P _G1-3 vHH #4 and the corresponding C3b overexpression strain CBS2612 P _G1-3 vHH C3b #13 were cultured in duplicate in a 1.8 L benchtop bioreactor (SR1500ODLS; Dasgip, Germany). did. For preculture, 100 mL of YPG medium containing 50 μg/mL ⁻¹ zeocin and 100 μg/mL ⁻¹ notheothricin (if applicable) in a 1 L shake flask was inoculated with 1.0 mL cryostoc and incubated at 180 rpm. , and incubated at 25°C for approximately 24 hours. Batch cultures were operated at a working volume of 0.6 L and inoculated at a starting OD ₆₀₀ of 0.4. The above medium was used for batch culture and continuous culture. In batch cultures, DO was maintained at 30% by automatic adjustment of stirring speed (400-1200 rpm) and air flow rate (9.5-30 sL/hr ^-1 ). For continuous culture, stirrer speed was set at 700 rpm and air flow rate was set at 30 sL/hr ⁻¹ . Throughout the process, the temperature was maintained at 30° C. and the pH at 5 by automatic addition of 12.5% NH ₄ OH. The medium was designed so that YDM reached a concentration of approximately 10 g/L in batch and continuous cultures. Continuous culture was started after a sudden spike in DO indicating the end of the batch. When μ=D=0.015/h ⁻¹ , the selected feed rate was 9 mL/h ⁻¹ and the culture volume was kept constant at 0.6 L. This was done by using a level sensor and automatically pumping additional culture from the reactor whenever the volume exceeded 0.6L. Samples were taken after 333 hours, corresponding to a change in reactor volume of 5 volumes, which was accepted as steady state conditions.

Yeast dry mass (YDM) and secreted recombinant proteins were analyzed at selected sampling points as described in Example 7b. Samples were also taken for analysis of transcript levels as described in Example 6a. Additionally, cell pellets were collected. This was made by centrifuging 1 mL of the culture at 16,000 g for 5 minutes, discarding the supernatant, and storing the pellet at -20°C. These were used for total protein measurements as described in Example 8c.

Table 22 shows the chemostat results described above. At this fixed, slow growth rate and even under steady-state conditions, titers were 1.75 times higher upon overexpression of TIF, while biomass concentration (YDM) was similar to the control host strain. Ta. Specific productivity increased 1.5-1.8 times in C3b overexpression lines compared to the host strain. This verifies that the positive effect of TIF overexpression on recombinant protein production seen in fed-batch cultures (Example 7) can also be achieved in continuous culture.

b) Effect of overexpression of translation factors on transcription levels in continuous culture.
To further elucidate the effects of C3b overexpression, transcript levels were measured using the procedure described in Example 6a. As described above, transcription levels were determined in S. Normalized to S. cerevisiae ACT1. In addition, they were normalized to the host strain (Reactor C050) to obtain the fold change in relative transcript levels of PpACT1 and vHH.

Overexpression of C3b resulted in a 1.40±0.04-fold higher relative transcript level of vHH. PpACT1 showed a similar fold change of 1.46±0.04. This confirms the results obtained in the screening and fed-batch cultures and shows that transcript levels are generally increased in cells overexpressing the selected translation initiation factors, independent of the culture mode applied. show.

c) Determination of total protein concentration.
To determine whether the effect of this increased transcript level was on the concentration of total cellular protein, the biuret method was used.

Briefly, the harvested cell pellet described in Example 8a was washed three times with water. They were then diluted with water and 8 mg/mL ⁻¹ YDM was added to each tube. 240 μL of cell suspension per sample was mixed with 125 μL of 3M NaOH and boiled at 99° C. for 5 minutes. After cooling, 125 μL of 2.5% CuSO4 was added and the samples were centrifuged at 16,000 g for 5 minutes. Of the obtained supernatant, 200 μL per tube was used for measurement with a Tecan Reader (Tecan Infinite M200) at a wavelength of 555 nm. For the calibration curve, dilutions of bovine serum albumin (albumin fraction V, for molecular biology ≥98%) (13, 12, 10, 8, 6, 4, 2, 1, and 0 g/L ⁻¹ ) were used. , processed in the same way as the samples.

Two reactors CBS2612 P _G1-3 vHH #4 with host strain produce 0.24 ± 0.00 mg protein per mg dry mass, while two reactors CBS2612 P _G1-3 vHH with overexpression strain C3b#13 produced 0.29±0.00 mg protein per mg dry mass. The 1.2-fold increase in total protein observed in the overexpression lines indicates that increased transcript levels result in increased total protein within the cell. However, the effect on recombinant POIs is significantly stronger (1.8-fold) than on overall cellular proteins (1.2-fold), indicating that during recombinant protein production, TIF of mRNPs is limited and their Our surprising finding that overexpression leads to higher productivity is again highlighted.

Example 9: Effect of overexpression of TIF on secretion of other model proteins.
To confirm the effect of overexpression of TIF, human serum albumin (HSA) was chosen as another model protein.

a) Generation of HSA-producing strains The expression cassette of P _{G1-3_HSA} was transformed into CBS2612 to generate HSA-producing strains as described in Example 1a. The resulting strains were screened as described in Example 2a and measured by microfluidic capillary electrophoresis (mCE) as described in Example 2c. Two HSA producing clones with different productivity were selected as hosts for TIF overexpression. CBS2612 _PG1-3 HSA#15 was selected as the average producing host strain, while CBS2612 _PG1-3 HSA#10 was selected as the high producing host strain. These two strains were rescreened in quadruplicate. The results can be seen in Table 23. In addition, the GCN of these strains was determined to explain differences in productivity. Determination of GCN was performed according to Example 1d.

The titers found in Table 23 represent the average of quadruplicate. High producers produce five times more than average producers, which correlates well with higher GCN. Both strains were used for overexpression of selected translation initiation factor constructs.

b) Generation of TIF overexpressing strains and their effect on HSA secretion A combination of overexpressing C3b was selected and tested in the two HSA producing strains described in Example 9a. Cloning was performed as described in Example 1, followed by screening and titering as described in Examples 2a and 2c.

Table 24 shows the screening results. Despite the difference in absolute HSA titers between the two production host strains, the effect of overexpression of TIF C3b is almost the same, with a 1.4-fold increase. This indicates that overexpression of TIF has the effect of increasing secretion of recombinant protein in high and average producing strains. In addition, the results in Table 24 show that overexpression of C3b increases recombinant protein secretion, not only for vHH but also for HSA, thus This reinforces the concept of a general positive effect of TIF overexpression on the production of transgenic proteins.

c) Fed-batch cultivation of HSA-producing strains overexpressing TIF The HSA host strain and the corresponding C3b-overexpressing strain were then used for fed-batch cultivation. Fed-batch cultivation was performed as described in Example 7. In this case, for linearly increasing glucose supply, the following equation was used: F [mL/h ⁻¹ ]=0.01*t+2. This resulted in an approximate growth rate of 0.029/h ^-1 at sampling point F9.

Regarding other model proteins and strains, as can be seen in Table 25, C3b overexpression increased the yield of secretion of recombinant HSA in both production host strains. In particular, the increase in secreted protein is much more pronounced in the high producing strain CBS2612 _PG1-3 HSA#10, with a 1.9-fold increase. Thereby, TIF of mRNPs poses a stronger bottleneck for cells with higher capacity for recombinant protein expression (e.g. higher transcription due to higher GCN and/or promoters with higher expression strength), and such It is concluded that cells further benefit from overexpression of TIF.

Claims (21)

A recombinant eukaryotic host cell expressing a gene of interest (GOI), wherein the gene is modified to enhance the expression of two or more genes encoding messenger ribonucleoprotein (mRNP) translation initiation factors (TIF genes). , wherein the TIF gene comprises at least a gene encoding eIF4A and a gene encoding eIF4G, and wherein the TIF gene comprises at least a gene encoding eIF4A and a gene encoding eIF4G; A recombinant eukaryotic host cell, wherein expression of at least one of the genes is under transcriptional control of a promoter different from the promoter controlling expression of said GOI. a) said eIF4A comprises at least 60% sequence identity to any one of SEQ ID NOs: 12-33;
b) The host cell of claim 1, wherein said eIF4G comprises at least 60% sequence identity to any one of SEQ ID NOs: 34-44. The TIF gene is
a) gene encoding eIF4E,
The host cell according to claim 1 or 2, further comprising any one or more of b) a gene encoding PAB1, or c) a gene encoding RLI1. a) said eIF4E comprises at least 60% sequence identity to any one of SEQ ID NOs: 1-11;
b) said PAB1 comprises at least 60% sequence identity to any one of SEQ ID NOs: 45-55;
c) The host cell of claim 3, wherein said RLI1 comprises at least 60% sequence identity to any one of SEQ ID NOs: 56-65. A host cell according to any one of claims 1 to 4, wherein one or more of the TIF genes is optimized to express the TIF gene in the host cell. At least a) genes encoding eIF4A and eIF4G,
b) genes encoding eIF4A, eIF4G, and eIF4E;
c) genes encoding eIF4A, eIF4G, eIF4E, and PAB1;
d) The host cell according to any one of claims 1 to 5, overexpressing genes encoding eIF4A, eIF4G and PAB1. 7. The host cell of claim 6, wherein the host cell is further engineered to overexpress a gene encoding RLI1. Said genetic modification is carried out from (i) one or more polynucleotides or portions thereof, or (ii) expression control sequences, preferably promoters, ribosome binding sites, transcription or translation initiation and termination sequences, enhancers and activator sequences. Preferably, one or more of said genetic modifications involve the incorporation of a heterologous polynucleotide or expression cassette into said host cell genome. The host cell according to any one of claims 1 to 7, comprising: the genetic modification comprises an increase in the number of the TIF genes or the number of expression cassettes containing the TIF genes, and/or a gain-of-function change in the TIF genes, resulting in an increase in the level or activity of the TIF genes; A host cell according to any one of claims 1 to 8. The host cell according to any one of claims 1 to 9, wherein the TIF gene is endogenous or heterologous to the host cell. a) an expression system for expressing one or more of said TIF genes in one or more heterologous TIF expression cassettes, each comprising one or more expression control sequences operably linked to said TIF gene; an expression system comprising;
b) a GOI expression cassette comprising a GOI and one or more expression control sequences operably linked to said GOI;
Any of claims 1 to 10, wherein the expression system of a) and the expression cassette of b) are engineered to express the respective TIF gene and GOI when culturing the host cell in cell culture. The host cell according to paragraph 1. a) at least one of said TIF expression cassettes comprises a constitutive promoter, and/or b) said GOI expression cassette comprises an inducible, derepressible, or otherwise regulatable promoter, or a constitutive promoter 12. The host cell of claim 11, comprising: a) Yeast cells of a genus selected from the group consisting of Pichia, Hansenula, Komagataella, Saccharomyces, Krivellomyces, Candida, Ogataea, Yarrowia, and Geotrichum (preferably Pichia pastoris, Komagataella phaffii, Komagataella pastoris, Komagataella pseudopastoris, Saccharomyces cerevisiae, Ogataea minuta, Kluyveromces lactis, Kluyveromes marxianus, Yarrowia lipolytica, or Hansenula polymorpha),
b) cells of filamentous fungi (e.g. Aspergillus awamori or Trichoderma reesei),
c) non-human primate, human, rodent, or bovine cells (e.g., mouse myeloma (NSO) cell line, Chinese hamster ovary (CHO) cell line, HT1080, H9, HepG2, MCF7, MDBKJurkat, MDCK, NIH3T3 , PC12, BHK (baby hamster kidney cells), VERO, SP2/0, YB2/0, Y0, C127, L cells, COS (COS1 and COS7, etc.), QC1-3, HEK-293, VERO, PER.C6, HeLA, EBI, EB2, EB3, oncolytic or hybridoma cell lines),
d) insect cells (e.g. Sf9, Mimic™ Sf9, Sf21, High Five (BT1-TN-5B1-4), or BT1-Ea88 cells),
e) algal cells (e.g. Amphora, Chlorella, Dunaliella, Chlorella, Chlamydomonas, Cyanobacteria, Nannochloropsis, Spirulina, or Ochromonas); or f) plant cells (e.g. , cells derived from monocotyledonous plants (preferably corn, rice, wheat, or hackberry), or cells derived from dicotyledonous plants (preferably cassava, potato, soybean, tomato, tobacco, alfalfa, Physcomitrella patens, or Arabidopsis). ), the host cell according to any one of claims 1 to 12. Any one of claims 1 to 13 comprising genetically engineering a host cell to include one or more of said TIF genes and a gene of interest (GOI) in one or more heterologous expression cassettes. A method for producing a host cell as described in Section. 15. A method for producing a protein of interest (POI) encoded by a gene of interest (GOI), wherein said production comprises a host cell according to claim 14 under conditions for producing said POI. A method carried out by culturing. the host cell is cultured in a culture medium under conditions to co-express one or more of the TIF genes and secrete the POI into the host cell culture; 16. The method of claim 15, wherein the method is recovered from a culture. the host cell at a level that increases the specific productivity (μg/g yeast dry mass (YDM)/hour) and/or volumetric productivity (μg/L/hour) of the host cell relative to the POI; 17. The method of claim 15 or 16, wherein the method is modified to co-express one or more of the TIF genes. The POI is a therapeutic or diagnostic agent, preferably an antigen binding protein, a therapeutic protein, an enzyme, a peptide, a protein antibiotic, a toxin fusion protein, a carbohydrate-protein conjugate, a structural protein, a regulatory protein, a vaccine antigen, a growth factor. 18. The method according to any one of claims 15 to 17, wherein the peptide, polypeptide, or protein is selected from the group consisting of , hormones, cytokines, process enzymes, and metabolic enzymes. A method of increasing the yield of a protein of interest (POI) of messenger ribonucleoprotein (mRNP) in cell culture when produced by a host cell expressing a gene of interest (GOI) encoding said POI. A method of increasing the expression of one or more TIF genes by co-expressing one or more heterologous expression cassettes. A polypeptide expression system comprising one or more heterologous expression cassettes expressing one or more TIF genes of messenger ribonucleoprotein (mRNP). 21. The expression of claim 20, comprising an expression cassette comprising a gene of interest (GOI) encoding a protein of interest (POI) and one or more expression control sequences operably linked to said GOI. system.